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1.
Development ; 151(20)2024 Oct 15.
Article in English | MEDLINE | ID: mdl-38950937

ABSTRACT

The capacity to regenerate lost tissues varies significantly among animals. Some phyla, such as the annelids, display substantial regenerating abilities, although little is known about the cellular mechanisms underlying the process. To precisely determine the origin, plasticity and fate of the cells participating in blastema formation and posterior end regeneration after amputation in the annelid Platynereis dumerilii, we developed specific tools to track different cell populations. Using these tools, we find that regeneration is partly promoted by a population of proliferative gut cells whose regenerative potential varies as a function of their position along the antero-posterior axis of the worm. Gut progenitors from anterior differentiated tissues are lineage restricted, whereas gut progenitors from the less differentiated and more proliferative posterior tissues are much more plastic. However, they are unable to regenerate the stem cells responsible for the growth of the worms. Those stem cells are of local origin, deriving from the cells present in the segment abutting the amputation plane, as are most of the blastema cells. Our results favour a hybrid and flexible cellular model for posterior regeneration in Platynereis relying on different degrees of cell plasticity.


Subject(s)
Cell Plasticity , Cell Proliferation , Polychaeta , Regeneration , Animals , Regeneration/physiology , Polychaeta/physiology , Polychaeta/cytology , Cell Plasticity/physiology , Stem Cells/cytology , Cell Differentiation/physiology , Annelida/physiology
2.
Cell Rep ; 42(1): 111967, 2023 01 31.
Article in English | MEDLINE | ID: mdl-36640345

ABSTRACT

Hox genes encode transcription factors that specify segmental identities along the anteroposterior body axis. These genes are organized in clusters, where their order corresponds to their activity along the body axis, a feature known as collinearity. In Drosophila, the BX-C cluster contains the three most posterior Hox genes, where their collinear activation incorporates progressive changes in histone modifications, chromatin architecture, and use of boundary elements and cis-regulatory regions. To dissect functional hierarchies, we compare chromatin organization in cell lines and larvae, with a focus on the Abd-B gene. Our work establishes the importance of the Fab-7 boundary for insulation between 3D domains carrying different histone modifications. Interestingly, we detect a non-canonical inversion of collinear chromatin dynamics at Abd-B, with the domain of active histone modifications progressively decreasing in size. This dynamic chromatin organization differentially activates the alternative promoters of the Abd-B gene, thereby expanding the possibilities for fine-tuning of transcriptional output.


Subject(s)
Drosophila Proteins , Drosophila , Animals , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Homeodomain Proteins/metabolism , Promoter Regions, Genetic/genetics , Regulatory Sequences, Nucleic Acid , Genes, Homeobox , Chromatin , Gene Expression Regulation, Developmental
3.
PLoS Genet ; 18(5): e1009782, 2022 05.
Article in English | MEDLINE | ID: mdl-35604932

ABSTRACT

The hallmarks of the alveolar subclass of rhabdomyosarcoma are chromosomal translocations that generate chimeric PAX3-FOXO1 or PAX7-FOXO1 transcription factors. Overexpression of either PAX-FOXO1s results in related cell transformation in animal models. Yet, in patients the two structural genetic aberrations they derived from are associated with distinct pathological manifestations. To assess the mechanisms underlying these differences, we generated isogenic fibroblast lines expressing either PAX-FOXO1 paralog. Mapping of their genomic recruitment using CUT&Tag revealed that the two chimeric proteins have distinct DNA binding preferences. In addition, PAX7-FOXO1 binding results in greater recruitment of the H3K27ac activation mark than PAX3-FOXO1 binding and is accompanied by greater transcriptional activation of neighbouring genes. These effects are associated with a PAX-FOXO1-specific alteration in the expression of genes regulating cell shape and the cell cycle. Consistently, PAX3-FOXO1 accentuates fibroblast cellular traits associated with contractility and surface adhesion and limits entry into S phase. In contrast, PAX7-FOXO1 drives cells to adopt an amoeboid shape, reduces entry into M phase, and causes increased DNA damage. Altogether, our results argue that the diversity of rhabdomyosarcoma manifestation arises, in part, from the divergence between the genomic occupancy and transcriptional activity of PAX3-FOXO1 and PAX7-FOXO1.


Subject(s)
Oncogene Proteins, Fusion , Paired Box Transcription Factors , Rhabdomyosarcoma, Alveolar , Animals , Cell Line , Cell Transformation, Neoplastic/genetics , Fibroblasts , Forkhead Box Protein O1/genetics , Forkhead Transcription Factors/genetics , Humans , Oncogene Proteins, Fusion/genetics , PAX3 Transcription Factor/genetics , PAX7 Transcription Factor/genetics , Paired Box Transcription Factors/genetics , Rhabdomyosarcoma/genetics , Rhabdomyosarcoma, Alveolar/genetics
4.
PLoS Genet ; 16(11): e1009164, 2020 11.
Article in English | MEDLINE | ID: mdl-33175861

ABSTRACT

The chromosome translocations generating PAX3-FOXO1 and PAX7-FOXO1 chimeric proteins are the primary hallmarks of the paediatric fusion-positive alveolar subtype of Rhabdomyosarcoma (FP-RMS). Despite the ability of these transcription factors to remodel chromatin landscapes and promote the expression of tumour driver genes, they only inefficiently promote malignant transformation in vivo. The reason for this is unclear. To address this, we developed an in ovo model to follow the response of spinal cord progenitors to PAX-FOXO1s. Our data demonstrate that PAX-FOXO1s, but not wild-type PAX3 or PAX7, trigger the trans-differentiation of neural cells into FP-RMS-like cells with myogenic characteristics. In parallel, PAX-FOXO1s remodel the neural pseudo-stratified epithelium into a cohesive mesenchyme capable of tissue invasion. Surprisingly, expression of PAX-FOXO1s, similar to wild-type PAX3/7, reduce the levels of CDK-CYCLIN activity and increase the fraction of cells in G1. Introduction of CYCLIN D1 or MYCN overcomes this PAX-FOXO1-mediated cell cycle inhibition and promotes tumour growth. Together, our findings reveal a mechanism that can explain the apparent limited oncogenicity of PAX-FOXO1 fusion transcription factors. They are also consistent with certain clinical reports indicative of a neural origin of FP-RMS.


Subject(s)
Cell Transdifferentiation/genetics , Cell Transformation, Neoplastic/genetics , Oncogene Proteins, Fusion/metabolism , Paired Box Transcription Factors/metabolism , Rhabdomyosarcoma, Alveolar/genetics , Animals , Biopsy , Chick Embryo , Child , Cyclin D1/genetics , Datasets as Topic , Disease Models, Animal , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , N-Myc Proto-Oncogene Protein/genetics , Neoplasm Invasiveness/genetics , Neural Stem Cells/pathology , Neural Tube/cytology , Oncogene Proteins, Fusion/genetics , PAX3 Transcription Factor/genetics , PAX3 Transcription Factor/metabolism , PAX7 Transcription Factor/genetics , PAX7 Transcription Factor/metabolism , Paired Box Transcription Factors/genetics , Rhabdomyosarcoma, Alveolar/pathology , S Phase/genetics
5.
Nucleic Acids Res ; 48(5): 2357-2371, 2020 03 18.
Article in English | MEDLINE | ID: mdl-31943068

ABSTRACT

The spatiotemporal expression of genes is controlled by enhancer sequences that bind transcription factors. Identifying the target genes of enhancers remains difficult because enhancers regulate gene expression over long genomic distances. To address this, we used an evolutionary approach to build two genome-wide maps of predicted enhancer-gene associations in the human and zebrafish genomes. Evolutionary conserved sequences were linked to their predicted target genes using PEGASUS, a bioinformatics method that relies on evolutionary conservation of synteny. The analysis of these maps revealed that the number of predicted enhancers linked to a gene correlate with its expression breadth. Comparison of both maps identified hundreds of putative vertebrate ancestral regulatory relationships from which we could determine that predicted enhancer-gene distances scale with genome size despite strong positional conservation. The two maps represent a resource for further studies, including the prioritization of sequence variants in whole genome sequence of patients affected by genetic diseases.


Subject(s)
Enhancer Elements, Genetic , Gene Expression Regulation, Developmental , Genetic Linkage , Transcription Factors/genetics , Animals , Base Sequence , Biological Evolution , Chromosome Mapping , Computational Biology/methods , Conserved Sequence , Embryo, Nonmammalian , Genome Size , Humans , Synteny , Transcription Factors/metabolism , Zebrafish
6.
PLoS Genet ; 14(8): e1007581, 2018 08.
Article in English | MEDLINE | ID: mdl-30080860

ABSTRACT

Cis-regulation plays an essential role in the control of gene expression, and is particularly complex and poorly understood for developmental genes, which are subject to multiple levels of modulation. In this study, we performed a global analysis of the cis-acting elements involved in the control of the zebrafish developmental gene krox20. krox20 encodes a transcription factor required for hindbrain segmentation and patterning, a morphogenetic process highly conserved during vertebrate evolution. Chromatin accessibility analysis reveals a cis-regulatory landscape that includes 6 elements participating in the control of initiation and autoregulatory aspects of krox20 hindbrain expression. Combining transgenic reporter analyses and CRISPR/Cas9-mediated mutagenesis, we assign precise functions to each of these 6 elements and provide a comprehensive view of krox20 cis-regulation. Three important features emerged. First, cooperation between multiple cis-elements plays a major role in the regulation. Cooperation can surprisingly combine synergy and redundancy, and is not restricted to transcriptional enhancer activity (for example, 4 distinct elements cooperate through different modes to maintain autoregulation). Second, several elements are unexpectedly versatile, which allows them to be involved in different aspects of control of gene expression. Third, comparative analysis of the elements and their activities in several vertebrate species reveals that this versatility is underlain by major plasticity across evolution, despite the high conservation of the gene expression pattern. These characteristics are likely to be of broad significance for developmental genes.


Subject(s)
Early Growth Response Protein 2/genetics , Gene Expression Regulation, Developmental , Rhombencephalon/metabolism , Zebrafish Proteins/genetics , Zebrafish/genetics , Amino Acid Sequence , Animals , CRISPR-Cas Systems , Chromatin/metabolism , Early Growth Response Protein 2/physiology , Enhancer Elements, Genetic , Evolution, Molecular , Genetic Loci , Morphogenesis/genetics , Transcriptional Activation , Zebrafish/embryology
7.
PLoS Genet ; 13(7): e1006903, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28749941

ABSTRACT

Developmental genes can harbour multiple transcriptional enhancers that act simultaneously or in succession to achieve robust and precise spatiotemporal expression. However, the mechanisms underlying cooperation between cis-acting elements are poorly documented, notably in vertebrates. The mouse gene Krox20 encodes a transcription factor required for the specification of two segments (rhombomeres) of the developing hindbrain. In rhombomere 3, Krox20 is subject to direct positive feedback governed by an autoregulatory enhancer, element A. In contrast, a second enhancer, element C, distant by 70 kb, is active from the initiation of transcription independent of the presence of the KROX20 protein. Here, using both enhancer knock-outs and investigations of chromatin organisation, we show that element C possesses a dual activity: besides its classical enhancer function, it is also permanently required in cis to potentiate the autoregulatory activity of element A, by increasing its chromatin accessibility. This work uncovers a novel, asymmetrical, long-range mode of cooperation between cis-acting elements that might be essential to avoid promiscuous activation of positive autoregulatory elements.


Subject(s)
Early Growth Response Protein 1/genetics , Enhancer Elements, Genetic , Regulatory Elements, Transcriptional/genetics , Rhombencephalon/growth & development , Animals , Body Patterning/genetics , Chromatin/genetics , Early Growth Response Protein 1/biosynthesis , Gene Expression Regulation, Developmental , Mice, Knockout , Mutation , Rhombencephalon/metabolism , Sequence Homology, Nucleic Acid
9.
Nat Genet ; 47(9): 1073-8, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26214589

ABSTRACT

Deciphering the ways in which somatic mutations and germline susceptibility variants cooperate to promote cancer is challenging. Ewing sarcoma is characterized by fusions between EWSR1 and members of the ETS gene family, usually EWSR1-FLI1, leading to the generation of oncogenic transcription factors that bind DNA at GGAA motifs. A recent genome-wide association study identified susceptibility variants near EGR2. Here we found that EGR2 knockdown inhibited proliferation, clonogenicity and spheroidal growth in vitro and induced regression of Ewing sarcoma xenografts. Targeted germline deep sequencing of the EGR2 locus in affected subjects and controls identified 291 Ewing-associated SNPs. At rs79965208, the A risk allele connected adjacent GGAA repeats by converting an interspaced GGAT motif into a GGAA motif, thereby increasing the number of consecutive GGAA motifs and thus the EWSR1-FLI1-dependent enhancer activity of this sequence, with epigenetic characteristics of an active regulatory element. EWSR1-FLI1 preferentially bound to the A risk allele, which increased global and allele-specific EGR2 expression. Collectively, our findings establish cooperation between a dominant oncogene and a susceptibility variant that regulates a major driver of Ewing sarcomagenesis.


Subject(s)
Bone Neoplasms/genetics , Early Growth Response Protein 2/genetics , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Protein c-fli-1/genetics , RNA-Binding Protein EWS/genetics , Sarcoma, Ewing/genetics , Animals , Base Sequence , Bone Neoplasms/pathology , Carotenoids/genetics , Cell Line, Tumor , Cell Proliferation , Gene Expression , Gene Expression Regulation, Neoplastic , Genetic Association Studies , Genetic Predisposition to Disease , Humans , Mice, SCID , Microsatellite Repeats , Molecular Sequence Data , Neoplasm Transplantation , Oxygenases/genetics , Polymorphism, Single Nucleotide , Quantitative Trait Loci , Sarcoma, Ewing/pathology , Tumor Burden
10.
Development ; 142(1): 185-95, 2015 Jan 01.
Article in English | MEDLINE | ID: mdl-25516974

ABSTRACT

Although many components of the genetic pathways that provide positional information during embryogenesis have been identified, it remains unclear how these signals are integrated to specify discrete tissue territories. Here, we investigate the molecular mechanisms underlying the formation of one of the hindbrain segments, rhombomere (r) 3, specified by the expression of the gene krox20. Dissecting krox20 transcriptional regulation has identified several input pathways: Hox paralogous 1 (PG1) factors, which both directly activate krox20 and indirectly repress it via Nlz factors, and the molecular components of an Fgf-dependent effector pathway. These different inputs are channelled through a single initiator enhancer element to shape krox20 initial transcriptional response: Hox PG1 and Nlz factors define the anterior-posterior extent of the enhancer's domain of activity, whereas Fgf signalling modulates the magnitude of activity in a spatially uniform manner. Final positioning of r3 boundaries requires interpretation of this initial pattern by a krox20 positive-feedback loop, orchestrated by another enhancer. Overall, this study shows how positional information provided by different patterning mechanisms is integrated through a gene regulatory network involving two cis-acting elements operating on the same gene, thus offering a comprehensive view of the delimitation of a territory.


Subject(s)
Body Patterning/genetics , Rhombencephalon/embryology , Rhombencephalon/metabolism , Zebrafish/embryology , Zebrafish/genetics , Animals , Enhancer Elements, Genetic/genetics , Gene Expression Regulation, Developmental , Models, Biological , Signal Transduction/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism
11.
Mol Syst Biol ; 9: 690, 2013.
Article in English | MEDLINE | ID: mdl-24061538

ABSTRACT

Although feedback loops are essential in development, their molecular implementation and precise functions remain elusive. Using enhancer knockout in mice, we demonstrate that a direct, positive autoregulatory loop amplifies and maintains the expression of Krox20, a transcription factor governing vertebrate hindbrain segmentation. By combining quantitative data collected in the zebrafish with biophysical modelling that accounts for the intrinsic stochastic molecular dynamics, we dissect the loop at the molecular level. We find that it underpins a bistable switch that turns a transient input signal into cell fate commitment, as we observe in single cell analyses. The stochasticity of the activation process leads to a graded input-output response until saturation is reached. Consequently, the duration and strength of the input signal controls the size of the hindbrain segments by modulating the distribution between the two cell fates. Moreover, segment formation is buffered from severe variations in input level. Finally, the progressive extinction of Krox20 expression involves a destabilization of the loop by repressor molecules. These mechanisms are of general significance for cell type specification and tissue patterning.


Subject(s)
Body Patterning/genetics , Early Growth Response Protein 1/genetics , Early Growth Response Protein 2/genetics , Feedback, Physiological , Gene Expression Regulation, Developmental , Rhombencephalon/cytology , Amino Acid Sequence , Animals , Animals, Genetically Modified , Cell Differentiation , Cell Proliferation , Chick Embryo , Early Growth Response Protein 1/metabolism , Early Growth Response Protein 2/metabolism , Embryo, Mammalian , Embryo, Nonmammalian , Enhancer Elements, Genetic , In Situ Hybridization , Mice , Molecular Sequence Data , Rhombencephalon/growth & development , Rhombencephalon/metabolism , Signal Transduction , Stochastic Processes , Transcription, Genetic , Zebrafish
12.
Endocrinology ; 154(1): 270-82, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23150495

ABSTRACT

In this study, we have investigated the expression and function of the transcription factor early growth response factor 2 (Egr2)/Krox20 in the developing anterior pituitary. Egr2 is initially expressed in all differentiating hormonal cells types, but its expression is mostly restricted to the somatotroph lineage after birth. Egr2 knockout results in anterior pituitary hypoplasia. However, the analysis of a conditional mutant demonstrates that this phenotype does not originate from a lack of Egr2 expression in the pituitary. Using an Egr2 allele driving a Cre-activable toxin gene, we performed a genetic ablation of Egr2-positive cells in the pituitary. During the postnatal period, this ablation leads to specific and progressive depletion of the somatotroph population, creating a novel model of early-onset isolated GH deficiency (GHD). Mutant animals were subjected to a complete metabolic analysis, revealing atypical and expected features. Consistent with an adult-onset isolated GHD model, mutant animals are hypoglycemic and display increased insulin sensitivity and glucose clearance. This latter phenotype is in contrast to the glucose intolerance observed in another early-onset GHD model. Surprisingly, increased insulin sensitivity is not accompanied by a modified balance between fat and lean tissues, but by reduced metabolic adaptability between glucose and lipid oxidation conditions. This suggests that the relationship between these metabolic features and insulin sensitivity should be reconsidered. In conclusion, our mutant may be a valuable genetic model with which to study the effects of long-term GH deficiency, in conditions of normal pancreatic function and unaffected balance between fat and glucose metabolism.


Subject(s)
Early Growth Response Protein 2/metabolism , Hypopituitarism/metabolism , Hypopituitarism/pathology , Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/metabolism , Alleles , Animals , Growth Hormone/blood , Immunohistochemistry , Insulin-Like Growth Factor I/metabolism , Male , Mice
13.
Glia ; 60(3): 393-403, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22379615

ABSTRACT

The analysis of the molecular mechanisms involved in the initial interaction between neurons and Schwann cells is a key issue in understanding the myelination process. We recently identified Cthrc1 (Collagen triple helix repeat containing 1) as a gene upregulated in Schwann cells upon interaction with the axon. Cthrc1 encodes a secreted protein previously shown to be involved in migration and proliferation in different cell types. We performed a functional analysis of Cthrc1 in Schwann cells by loss-of- and gain-of-function approaches using RNA interference knockdown in cell culture and a transgenic mouse line that overexpresses the gene. This work establishes that Cthrc1 enhances Schwann cell proliferation but prevents myelination. In particular, time-course analysis of myelin formation intransgenic animals reveals that overexpression of Cthrc1 in Schwann cells leads to a delay in myelin formation with cells maintaining a proliferative state. Our data, therefore, demonstrate that Cthrc1 plays a negative regulatory role, fine-tuning the onset of peripheral myelination.


Subject(s)
Extracellular Matrix Proteins/metabolism , Gene Expression Regulation/genetics , Schwann Cells/metabolism , Animals , Bromodeoxyuridine/metabolism , Cell Movement/drug effects , Cell Movement/genetics , Cell Proliferation , Cell Survival/drug effects , Cell Survival/genetics , Early Growth Response Protein 2/genetics , Embryo, Mammalian , Extracellular Matrix Proteins/genetics , Ganglia, Spinal/cytology , Gene Expression Regulation/drug effects , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Mice , Mice, Transgenic , Microscopy, Electron , Myelin Basic Protein/metabolism , RNA, Small Interfering/pharmacology , Rats , Rats, Sprague-Dawley , Schwann Cells/ultrastructure , Time Factors , Transfection
14.
J Biol Chem ; 286(7): 5855-67, 2011 Feb 18.
Article in English | MEDLINE | ID: mdl-21173153

ABSTRACT

The molecules involved in vertebrate tendon formation during development remain largely unknown. To date, only two DNA-binding proteins have been identified as being involved in vertebrate tendon formation, the basic helix-loop-helix transcription factor Scleraxis and, recently, the Mohawk homeobox gene. We investigated the involvement of the early growth response transcription factors Egr1 and Egr2 in vertebrate tendon formation. We established that Egr1 and Egr2 expression in tendon cells was correlated with the increase of collagen expression during tendon cell differentiation in embryonic limbs. Vertebrate tendon differentiation relies on a muscle-derived FGF (fibroblast growth factor) signal. FGF4 was able to activate the expression of Egr genes and that of the tendon-associated collagens in chick limbs. Egr gene misexpression experiments using the chick model allowed us to establish that either Egr gene has the ability to induce de novo expression of the reference tendon marker scleraxis, the main tendon collagen Col1a1, and other tendon-associated collagens Col3a1, Col5a1, Col12a1, and Col14a1. Mouse mutants for Egr1 or Egr2 displayed reduced amounts of Col1a1 transcripts and a decrease in the number of collagen fibrils in embryonic tendons. Moreover, EGR1 and EGR2 trans-activated the mouse Col1a1 proximal promoter and were recruited to the tendon regulatory regions of this promoter. These results identify EGRs as novel DNA-binding proteins involved in vertebrate tendon differentiation by regulating type I collagen production.


Subject(s)
Cell Differentiation/physiology , Early Growth Response Protein 1/metabolism , Early Growth Response Protein 2/metabolism , Embryo, Mammalian/embryology , Tendons/embryology , Animals , Avian Proteins/biosynthesis , Avian Proteins/genetics , Basic Helix-Loop-Helix Transcription Factors/biosynthesis , Basic Helix-Loop-Helix Transcription Factors/genetics , Chick Embryo , Chickens , Collagen/biosynthesis , Collagen/genetics , Early Growth Response Protein 1/genetics , Early Growth Response Protein 2/genetics , Embryo, Mammalian/cytology , Fibroblast Growth Factor 4/genetics , Fibroblast Growth Factor 4/metabolism , Mice , Mice, Knockout , Tendons/cytology
15.
Development ; 138(2): 317-26, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21177344

ABSTRACT

Vertebrate hindbrain segmentation is an evolutionarily conserved process that involves a complex interplay of transcription factors and signalling pathways. Fibroblast growth factor (FGF) signalling plays a major role, notably by controlling the expression of the transcription factor Krox20 (Egr2), which is required for the formation and specification of two segmental units: rhombomeres (r) 3 and 5. Here, we explore the molecular mechanisms downstream of FGF signalling and the function of Sprouty 4 (Spry4), a negative-feedback regulator of this pathway, in zebrafish. We show that precise modulation of FGF signalling by Spry4 is required to determine the appropriate onset of krox20 transcription in r3 and r5 and, ultimately, rhombomere size in the r3-r5 region. FGF signalling acts by modulating the activity of krox20 initiator enhancer elements B and C; in r5, we show that this regulation is mediated by direct binding of the transcription factor MafB to element B. By contrast, FGF signalling does not control the krox20 autoregulatory element A, which is responsible for amplification and maintenance of krox20 expression. Therefore, early krox20 transcription sets the blueprint for r3-r5 patterning. This work illustrates the necessity for fine-tuning in a common and fundamental patterning process, based on a bistable cell-fate choice involving the coupling of an extracellular gradient with a positive-feedback loop. In this mode of patterning, precision and robustness can be achieved by the introduction of a negative-feedback loop, which, in the hindbrain, is mediated by Spry4.


Subject(s)
Early Growth Response Protein 2/genetics , Nerve Tissue Proteins/metabolism , Rhombencephalon/embryology , Rhombencephalon/metabolism , Zebrafish Proteins/genetics , Zebrafish/embryology , Zebrafish/metabolism , Animals , Avian Proteins/genetics , Avian Proteins/metabolism , Base Sequence , Binding Sites/genetics , Body Patterning/genetics , Body Patterning/physiology , Chick Embryo , DNA Primers/genetics , Enhancer Elements, Genetic , Feedback, Physiological , Fibroblast Growth Factors/metabolism , Gene Expression Regulation, Developmental , MafB Transcription Factor/genetics , MafB Transcription Factor/metabolism , Multigene Family , Nerve Tissue Proteins/genetics , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction , Transcription, Genetic , Zebrafish/genetics , Zebrafish Proteins/metabolism
16.
J Biol Chem ; 284(16): 10831-40, 2009 Apr 17.
Article in English | MEDLINE | ID: mdl-19218566

ABSTRACT

The zinc finger transcription factor Krox20 plays an essential role in the vertebrate hindbrain segmentation process. It positively or negatively controls a large variety of other regulatory genes, coordinating delimitation of segmental territories, specification of their identity, and maintenance of their integrity. We have investigated the molecular mechanisms of Krox20 transcriptional control by performing a detailed structure-function analysis of the protein in the developing chick hindbrain. This revealed an unsuspected diversity in the modes of action of a transcription factor in a single tissue, since regulation of each of the five tested target genes requires different parts of the protein and/or presumably different co-factors. The multiplicity of Krox20 functions might rely on this diversity. Investigation of known Krox20 co-factors was initiated in relation to this analysis. Nab was shown to act as a negative feedback modulator of the different Krox20 activating functions in the hindbrain. HCF-1 was found to bind to a Krox20 N-terminal region, which was shown to rely on multiple elements, including acidic domains, to convey Nab activation and Krox20 autoregulation.


Subject(s)
Early Growth Response Protein 2/metabolism , Gene Expression Regulation, Developmental , Rhombencephalon/embryology , Transcription, Genetic , Animals , COS Cells , Cell Nucleus/metabolism , Chlorocebus aethiops , Early Growth Response Protein 2/genetics , Host Cell Factor C1/genetics , Host Cell Factor C1/metabolism , Mice , Morphogenesis , Mutation , Repressor Proteins/genetics , Repressor Proteins/metabolism , Rhombencephalon/anatomy & histology
17.
Dev Biol ; 327(2): 566-77, 2009 Mar 15.
Article in English | MEDLINE | ID: mdl-19152797

ABSTRACT

Patterning of the vertebrate hindbrain involves a segmentation process leading to the formation of seven rhombomeres along the antero-posterior axis. While recent studies have shed light on the mechanisms underlying progressive subdivision of the posterior hindbrain into individual rhombomeres, the early events involved in anterior hindbrain patterning are still largely unknown. In this paper we demonstrate that two zebrafish Iroquois transcription factors, Irx7 and Irx1b, are required for the proper formation and specification of rhombomeres 1 to 4 and, in particular, for krox20 activation in r3. We also show that Irx7 functionally interacts with Meis factors to activate the expression of anterior hindbrain markers, such as hoxb1a, hoxa2 and krox20, ectopically in the anterior neural plate. Then, focusing on krox20 expression, we show that the effect of Irx7 and Meis1.1 is mediated by element C, a conserved cis-regulatory element involved in krox20 activation in the hindbrain. Together, our data point to an essential function of Iroquois transcription factors in krox20 activation and, more generally, in anterior hindbrain specification.


Subject(s)
Body Patterning/physiology , Early Growth Response Protein 2/metabolism , Homeodomain Proteins/metabolism , Rhombencephalon , Transcription Factors/metabolism , Zebrafish Proteins/metabolism , Zebrafish , Animals , Biomarkers/metabolism , Early Growth Response Protein 2/genetics , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , In Situ Hybridization , Myeloid Ecotropic Viral Integration Site 1 Protein , Neural Plate/anatomy & histology , Neural Plate/physiology , Regulatory Elements, Transcriptional , Rhombencephalon/anatomy & histology , Rhombencephalon/embryology , Transcription Factors/genetics , Zebrafish/anatomy & histology , Zebrafish/embryology , Zebrafish/metabolism , Zebrafish Proteins/genetics
18.
Development ; 135(20): 3369-78, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18787068

ABSTRACT

The morphogenesis of the vertebrate hindbrain involves the generation of metameric units called rhombomeres (r), and Krox20 encodes a transcription factor that is expressed in r3 and r5 and plays a major role in this segmentation process. Our knowledge of the basis of Krox20 regulation in r3 is rather confusing, especially concerning the involvement of Hox factors. To investigate this issue, we studied one of the Krox20 hindbrain cis-regulatory sequences, element C, which is active in r3-r5 and which is the only initiator element in r3. We show that element C contains multiple binding sites for Meis and Hox/Pbx factors and that these proteins synergize to activate the enhancer. Mutation of these binding sites allowed us to establish that Krox20 is under the direct transcriptional control of both Meis (presumably Meis2) and Hox/Pbx factors in r3. Furthermore, our data indicate that element C functions according to multiple modes, in Meis-independent or -dependent manners and with different Hox proteins, in r3 and r5. Finally, we show that the Hoxb1 and Krox20 expression domains transiently overlap in prospective r3, and that Hoxb1 binds to element C in vivo, supporting a cell-autonomous involvement of Hox paralogous group 1 proteins in Krox20 regulation. Altogether, our data clarify the molecular mechanisms of an essential step in hindbrain patterning. We propose a model for the complex regulation of Krox20, involving a novel mode of initiation, positive and negative controls by Hox proteins, and multiple direct and indirect autoregulatory loops.


Subject(s)
Body Patterning , Early Growth Response Protein 2/metabolism , Enhancer Elements, Genetic , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Rhombencephalon/embryology , Animals , Early Growth Response Protein 2/genetics , Embryo, Mammalian , Gene Expression Regulation, Developmental , In Situ Hybridization , Mice , Mice, Transgenic , Models, Biological , Rhombencephalon/metabolism , Transcription, Genetic
19.
J Neurosci ; 28(23): 5891-900, 2008 Jun 04.
Article in English | MEDLINE | ID: mdl-18524893

ABSTRACT

Krox20/Egr2 is a zinc finger transcription factor that plays essential roles in several developmental processes, including peripheral nervous system myelination by Schwann cells, where it acts as a master gene regulator. Krox20 is known to interact with cofactors of the Nab family and a mutation affecting isoleucine 268, which prevents this interaction, has been shown to result in congenital hypomyelinating neuropathy in humans. To further investigate the role of this interaction, we have introduced such a mutation, Krox20(I268F), in the mouse germ line. Clinical, immunohistochemical, and ultrastructural analyses of the homozygous mutants reveal that they develop a severe hypomyelination phenotype that mimics the human syndrome. Furthermore, a time-course analysis of the disease indicates that it follows a biphasic evolution, the hypomyelination phase being followed by a dramatic demyelination. Although for the regulation of most analyzed Krox20 target genes the mutation behaves as a loss of function, this is not the case for a few of them. This differential effect indicates that the molecular function of the Krox20-Nab interaction is target dependent and might explain the degradation of the residual myelin, because of imbalances in its composition. In conclusion, this work provides a novel and useful model for severe human peripheral neuropathies.


Subject(s)
Amino Acid Substitution/genetics , Early Growth Response Protein 2/genetics , Neoplasm Proteins/genetics , Peripheral Nervous System Diseases/genetics , Repressor Proteins/genetics , Animals , COS Cells , Cells, Cultured , Chlorocebus aethiops , Early Growth Response Protein 2/metabolism , Female , Male , Mice , Mice, Mutant Strains , Neoplasm Proteins/metabolism , Nerve Fibers, Myelinated/pathology , Nerve Fibers, Myelinated/physiology , Peripheral Nervous System Diseases/metabolism , Peripheral Nervous System Diseases/pathology , Protein Binding/genetics , Repressor Proteins/metabolism , Time Factors
20.
Dev Biol ; 309(2): 344-57, 2007 Sep 15.
Article in English | MEDLINE | ID: mdl-17669392

ABSTRACT

The homeodomain transcription factor vHNF1 plays an essential role in the patterning of the caudal segmented hindbrain, where it participates in the definition of the boundary between rhombomeres (r) 4 and 5 and in the specification of the identity of r5 and r6. Understanding the molecular basis of vHnf1 own expression therefore constitutes an important issue to decipher the regulatory network governing hindbrain patterning. We have identified a highly conserved 800-bp enhancer element located in the fourth intron of vHnf1 and whose activity recapitulates vHnf1 neural expression in transgenic mice. Functional analysis of this enhancer revealed that it contains two types of essential motifs, a retinoic acid response element and two half T-MARE sites, indicating that it integrates direct inputs from the retinoic acid signaling cascade and MAF-related factors. Our data suggest that MAFB, which is itself regulated by vHNF1, acts as a positive modulator of vHnf1 in r5 and r6, whereas another MAF-related factor is absolutely required for the expression of vHnf1 in both the hindbrain and the spinal cord. We propose a model accounting for the initiation and maintenance phases of vHnf1 expression and for the establishment of the r4/r5 boundary, based on cooperative contributions of Maf factors and retinoic acid signaling.


Subject(s)
Hepatocyte Nuclear Factor 1-beta/metabolism , MafB Transcription Factor/physiology , Neural Tube/metabolism , Signal Transduction , Spinal Cord/metabolism , Tretinoin/physiology , Animals , Base Sequence , Enhancer Elements, Genetic , Gene Expression Regulation, Developmental , Hepatocyte Nuclear Factor 1-beta/genetics , Mice , Mice, Transgenic , Molecular Sequence Data , Neural Tube/embryology , Response Elements , Rhombencephalon/embryology , Rhombencephalon/metabolism , Spinal Cord/embryology
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