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1.
Biomedicines ; 12(4)2024 Apr 10.
Article in English | MEDLINE | ID: mdl-38672195

ABSTRACT

RASopathies, a group of neurodevelopmental congenital disorders stemming from mutations in the RAS/MAPK pathway, present a unique opportunity to delve into the intricacies of complex neurological disorders. Afflicting approximately one in a thousand newborns, RASopathies manifest as abnormalities across multiple organ systems, with a pronounced impact on the central and peripheral nervous system. In the pursuit of understanding RASopathies' neurobiology and establishing phenotype-genotype relationships, in vivo non-mammalian models have emerged as indispensable tools. Species such as Danio rerio, Drosophila melanogaster, Caenorhabditis elegans, Xenopus species and Gallus gallus embryos have proven to be invaluable in shedding light on the intricate pathways implicated in RASopathies. Despite some inherent weaknesses, these genetic models offer distinct advantages over traditional rodent models, providing a holistic perspective on complex genetics, multi-organ involvement, and the interplay among various pathway components, offering insights into the pathophysiological aspects of mutations-driven symptoms. This review underscores the value of investigating the genetic basis of RASopathies for unraveling the underlying mechanisms contributing to broader neurological complexities. It also emphasizes the pivotal role of non-mammalian models in serving as a crucial preliminary step for the development of innovative therapeutic strategies.

2.
Emerg Top Life Sci ; 7(4): 423-437, 2023 Dec 18.
Article in English | MEDLINE | ID: mdl-38087891

ABSTRACT

Neurulation is a critical step in early embryonic development, giving rise to the neural tube, the primordium of the central nervous system in amniotes. Understanding this complex, multi-scale, multi-tissue morphogenetic process is essential to provide insights into normal development and the etiology of neural tube defects. Innovations in tissue engineering have fostered the generation of pluripotent stem cell-based in vitro models, including organoids, that are emerging as unique tools for delving into neurulation mechanisms, especially in the context of human development. Each model captures specific aspects of neural tube morphogenesis, from epithelialization to neural tissue elongation, folding and cavitation. In particular, the recent models of human and mouse trunk morphogenesis, such as gastruloids, that form a spinal neural plate-like or neural tube-like structure are opening new avenues to study normal and pathological neurulation. Here, we review the morphogenetic events generating the neural tube in the mammalian embryo and questions that remain unanswered. We discuss the advantages and limitations of existing in vitro models of neurulation and possible future technical developments.


Subject(s)
Neural Tube Defects , Neurulation , Mice , Animals , Humans , Neurulation/physiology , Neural Tube , Neural Plate , Stem Cells , Mammals
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.
Development ; 148(6)2021 03 29.
Article in English | MEDLINE | ID: mdl-33782043

ABSTRACT

Rostro-caudal patterning of vertebrates depends on the temporally progressive activation of HOX genes within axial stem cells that fuel axial embryo elongation. Whether the pace of sequential activation of HOX genes, the 'HOX clock', is controlled by intrinsic chromatin-based timing mechanisms or by temporal changes in extrinsic cues remains unclear. Here, we studied HOX clock pacing in human pluripotent stem cell-derived axial progenitors differentiating into diverse spinal cord motor neuron subtypes. We show that the progressive activation of caudal HOX genes is controlled by a dynamic increase in FGF signaling. Blocking the FGF pathway stalled induction of HOX genes, while a precocious increase of FGF, alone or with GDF11 ligand, accelerated the HOX clock. Cells differentiated under accelerated HOX induction generated appropriate posterior motor neuron subtypes found along the human embryonic spinal cord. The pacing of the HOX clock is thus dynamically regulated by exposure to secreted cues. Its manipulation by extrinsic factors provides synchronized access to multiple human neuronal subtypes of distinct rostro-caudal identities for basic and translational applications.This article has an associated 'The people behind the papers' interview.


Subject(s)
Circadian Clocks , Homeodomain Proteins/metabolism , Motor Neurons/metabolism , Pluripotent Stem Cells/metabolism , Benzamides/pharmacology , Bone Morphogenetic Proteins/genetics , Bone Morphogenetic Proteins/metabolism , Bone Morphogenetic Proteins/pharmacology , Cell Differentiation , Circadian Clocks/drug effects , Diphenylamine/analogs & derivatives , Diphenylamine/pharmacology , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Embryonic Development , Fibroblast Growth Factors/antagonists & inhibitors , Fibroblast Growth Factors/metabolism , Fibroblast Growth Factors/pharmacology , Gene Expression Regulation, Developmental , Growth Differentiation Factors/genetics , Growth Differentiation Factors/metabolism , Growth Differentiation Factors/pharmacology , Homeodomain Proteins/genetics , Humans , Motor Neurons/cytology , Pluripotent Stem Cells/cytology , Pyrimidines/pharmacology , Signal Transduction/drug effects , Spinal Cord/metabolism
5.
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
6.
Elife ; 92020 02 27.
Article in English | MEDLINE | ID: mdl-32105214

ABSTRACT

The establishment of separated pulmonary and systemic circulation in vertebrates, via cardiac outflow tract (OFT) septation, is a sensitive developmental process accounting for 10% of all congenital anomalies. Neural Crest Cells (NCC) colonising the heart condensate along the primitive endocardial tube and force its scission into two tubes. Here, we show that NCC aggregation progressively decreases along the OFT distal-proximal axis following a BMP signalling gradient. Dullard, a nuclear phosphatase, tunes the BMP gradient amplitude and prevents NCC premature condensation. Dullard maintains transcriptional programs providing NCC with mesenchymal traits. It attenuates the expression of the aggregation factor Sema3c and conversely promotes that of the epithelial-mesenchymal transition driver Twist1. Altogether, Dullard-mediated fine-tuning of BMP signalling ensures the timed and progressive zipper-like closure of the OFT by the NCC and prevents the formation of a heart carrying the congenital abnormalities defining the tetralogy of Fallot.


Subject(s)
Myocardium/cytology , Neural Crest/cytology , Phosphoprotein Phosphatases/physiology , Smad1 Protein/metabolism , Smad5 Protein/metabolism , Smad8 Protein/metabolism , Animals , Gene Deletion , Gene Expression Regulation, Developmental , Heart/embryology , Mice , Myocardium/metabolism , Phosphoprotein Phosphatases/genetics , Signal Transduction , Smad1 Protein/genetics , Smad5 Protein/genetics , Smad8 Protein/genetics , Tetralogy of Fallot/prevention & control
7.
Nat Cell Biol ; 21(11): 1334-1345, 2019 11.
Article in English | MEDLINE | ID: mdl-31685991

ABSTRACT

It is well established that haematopoietic stem and progenitor cells (HSPCs) are generated from a transient subset of specialized endothelial cells termed haemogenic, present in the yolk sac, placenta and aorta, through an endothelial-to-haematopoietic transition (EHT). HSPC generation via EHT is thought to be restricted to the early stages of development. By using experimental embryology and genetic approaches in birds and mice, respectively, we document here the discovery of a bone marrow haemogenic endothelium in the late fetus/young adult. These cells are capable of de novo producing a cohort of HSPCs in situ that harbour a very specific molecular signature close to that of aortic endothelial cells undergoing EHT or their immediate progenies, i.e., recently emerged HSPCs. Taken together, our results reveal that HSPCs can be generated de novo past embryonic stages. Understanding the molecular events controlling this production will be critical for devising innovative therapies.


Subject(s)
Bone Marrow Cells/metabolism , Cell Lineage/genetics , Gene Expression Regulation, Developmental , Hemangioblasts/metabolism , Hematopoietic Stem Cells/metabolism , Animals , Animals, Genetically Modified , Aorta/cytology , Aorta/metabolism , Bone Marrow Cells/cytology , Cell Differentiation , Chickens , Embryo, Mammalian , Embryo, Nonmammalian , Female , Fetus , Gene Expression Profiling , Gene Regulatory Networks , Hemangioblasts/cytology , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Heterozygote , Homozygote , Male , Mice , Pregnancy , Yolk Sac/cytology , Yolk Sac/growth & development , Yolk Sac/metabolism
8.
Development ; 146(14)2019 07 25.
Article in English | MEDLINE | ID: mdl-31239243

ABSTRACT

Bone morphogenetic proteins (BMPs) are secreted regulators of cell fate in several developing tissues. In the embryonic spinal cord, they control the emergence of the neural crest, roof plate and distinct subsets of dorsal interneurons. Although a gradient of BMP activity has been proposed to determine cell type identity in vivo, whether this is sufficient for pattern formation in vitro is unclear. Here, we demonstrate that exposure to BMP4 initiates distinct spatial dynamics of BMP signalling within the self-emerging epithelia of both mouse and human pluripotent stem cell-derived spinal organoids. The pattern of BMP signalling results in the stereotyped spatial arrangement of dorsal neural tube cell types, and concentration, timing and duration of BMP4 exposure modulate these patterns. Moreover, differences in the duration of competence time-windows between mouse and human account for the species-specific tempo of neural differentiation. Together, this study describes efficient methods for generating patterned subsets of dorsal interneurons in spinal organoids and supports the conclusion that graded BMP activity orchestrates the spatial organization of the dorsal neural tube cellular diversity in mouse and human.


Subject(s)
Bone Morphogenetic Protein 4/physiology , Cell Differentiation/genetics , Organoids/physiology , Smad Proteins/metabolism , Spine/cytology , Animals , Cell Lineage/genetics , Cells, Cultured , Embryo, Mammalian , Gene Expression Regulation, Developmental , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/physiology , Interneurons/cytology , Interneurons/physiology , Mice , Neural Crest/cytology , Neural Crest/physiology , Neural Tube/cytology , Neural Tube/embryology , Neurons/cytology , Neurons/physiology , Organoids/cytology , Signal Transduction/genetics , Smad Proteins/genetics
9.
Development ; 146(4)2019 02 20.
Article in English | MEDLINE | ID: mdl-30760481

ABSTRACT

Specification of neurons in the spinal cord relies on extrinsic and intrinsic signals, which in turn are interpreted by expression of transcription factors. V2 interneurons develop from the ventral aspects of the spinal cord. We report here a novel neuronal V2 subtype, named V2s, in zebrafish embryos. Formation of these neurons depends on the transcription factors sox1a and sox1b. They develop from common gata2a- and gata3-dependent precursors co-expressing markers of V2b and V2s interneurons. Chemical blockage of Notch signalling causes a decrease in V2s and an increase in V2b cells. Our results are consistent with the existence of at least two types of precursor arranged in a hierarchical manner in the V2 domain. V2s neurons grow long ipsilateral descending axonal projections with a short branch at the ventral midline. They acquire a glycinergic neurotransmitter type during the second day of development. Unilateral ablation of V2s interneurons causes a delay in touch-provoked escape behaviour, suggesting that V2s interneurons are involved in fast motor responses.


Subject(s)
Gene Expression Regulation, Developmental , Interneurons/metabolism , Motor Neurons/metabolism , SOXB1 Transcription Factors/metabolism , Spinal Cord/metabolism , Zebrafish/embryology , Animals , Behavior, Animal , GATA2 Transcription Factor/metabolism , Genotype , Glycine/chemistry , Green Fluorescent Proteins/metabolism , Homeodomain Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Transgenic , Mutation , Receptors, Notch/metabolism , Signal Transduction , Species Specificity , Spinal Cord/embryology , Zebrafish/metabolism , Zebrafish Proteins/metabolism
10.
Dev Biol ; 432(1): 24-33, 2017 12 01.
Article in English | MEDLINE | ID: mdl-28625870

ABSTRACT

Transcription factors are key orchestrators of the emergence of neuronal diversity within the developing spinal cord. As such, the two paralogous proteins Pax3 and Pax7 regulate the specification of progenitor cells within the intermediate neural tube, by defining a neat segregation between those fated to form motor circuits and those involved in the integration of sensory inputs. To attain insights into the molecular means by which they control this process, we have performed detailed phenotypic analyses of the intermediate spinal interneurons (IN), namely the dI6, V0D, V0VCG and V1 populations in compound null mutants for Pax3 and Pax7. This has revealed that the levels of Pax3/7 proteins determine both the dorso-ventral extent and the number of cells produced in each subpopulation; with increasing levels leading to the dorsalisation of their fate. Furthermore, thanks to the examination of mutants in which Pax3 transcriptional activity is skewed either towards repression or activation, we demonstrate that this cell diversification process is mainly dictated by Pax3/7 ability to repress gene expression. Consistently, we show that Pax3 and Pax7 inhibit the expression of Dbx1 and of its repressor Prdm12, fate determinants of the V0 and V1 interneurons, respectively. Notably, we provide evidence for the activity of several cis-regulatory modules of Dbx1 to be sensitive to Pax3 and Pax7 transcriptional activity levels. Altogether, our study provides insights into how the redundancy within a TF family, together with discrete dynamics of expression profiles of each member, are exploited to generate cellular diversity. Furthermore, our data supports the model whereby cell fate choices in the neural tube do not rely on binary decisions but rather on inhibition of multiple alternative fates.


Subject(s)
Homeodomain Proteins/physiology , Interneurons/physiology , Nerve Tissue Proteins/physiology , PAX3 Transcription Factor/physiology , PAX7 Transcription Factor/physiology , Spinal Cord/cytology , Animals , Cell Differentiation/physiology , Chick Embryo , Gene Expression Regulation, Developmental , Interneurons/cytology , Mice , Neural Tube/physiology , Spinal Cord/embryology , Stem Cells/cytology , Stem Cells/physiology
11.
Semin Cell Dev Biol ; 44: 75-86, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26400366

ABSTRACT

Over the past two decades, Pax proteins have received a lot of attention from researchers working on the generation and assembly of neural circuits during vertebrate development. Through tissue or cell based phenotypic analyses, or more recently using genome-wide approaches, they have highlighted the pleiotropic functions of Pax proteins during neurogenesis. This review discusses the wide range of molecular and cellular mechanisms by which these transcription factors control in time and space the number and identity of neurons produced during development. We first focus on the position of Pax proteins within gene regulatory networks that generate patterns of cellular differentiation within the central nervous system. Next, the architecture of Pax-linked regulatory loops that provide a tempo of differentiation to progenitor cells is presented. Finally, we examine the molecular foundations providing a "multitasking" property to Pax proteins. Amongst the Pax factors that are expressed within the developing nervous system, Pax6 is the most extensively studied and thus holds a dominant position in this article.


Subject(s)
Gene Regulatory Networks , Neurons/physiology , Paired Box Transcription Factors/genetics , Animals , Cell Differentiation/genetics , Humans , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neural Stem Cells/physiology , Neurons/cytology , Neurons/metabolism , Paired Box Transcription Factors/metabolism
12.
Development ; 141(8): 1726-36, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24715462

ABSTRACT

Dorsal spinal neurogenesis is orchestrated by the combined action of signals secreted from the roof plate organizer and a downstream transcriptional cascade. Within this cascade, Msx1 and Msx2, two homeodomain transcription factors (TFs), are induced earlier than bHLH neuralizing TFs. Whereas bHLH TFs have been shown to specify neuronal cell fate, the function of Msx genes remains poorly defined. We describe dramatic alterations of neuronal patterning in Msx1/Msx2 double-mutant mouse embryos. The most dorsal spinal progenitor pool fails to express the bHLH neuralizing TF Atoh1, which results in a lack of Lhx2-positive and Barhl2-positive dI1 interneurons. Neurog1 and Ascl1 expression territories are dorsalized, leading to ectopic dorsal differentiation of dI2 and dI3 interneurons. In proportion, the amount of Neurog1-expressing progenitors appears unaffected, whereas the number of Ascl1-positive cells is increased. These defects occur while BMP signaling is still active in the Msx1/Msx2 mutant embryos. Cell lineage analysis and co-immunolabeling demonstrate that Atoh1-positive cells derive from progenitors expressing both Msx1 and Msx2. In vitro, Msx1 and Msx2 proteins activate Atoh1 transcription by specifically interacting with several homeodomain binding sites in the Atoh1 3' enhancer. In vivo, Msx1 and Msx2 are required for Atoh1 3' enhancer activity and ChIP experiments confirm Msx1 binding to this regulatory sequence. These data support a novel function of Msx1 and Msx2 as transcriptional activators. Our study provides new insights into the transcriptional control of spinal cord patterning by BMP signaling, with Msx1 and Msx2 acting upstream of Atoh1.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Gene Expression Regulation, Developmental , Homeodomain Proteins/metabolism , MSX1 Transcription Factor/metabolism , Spinal Cord/metabolism , Animals , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/metabolism , Binding Sites , Bone Morphogenetic Proteins/metabolism , Cell Differentiation/genetics , Embryo, Mammalian/metabolism , Enhancer Elements, Genetic/genetics , Interneurons/cytology , Interneurons/metabolism , Mice , Molecular Sequence Data , Mutation/genetics , Protein Binding/genetics , Signal Transduction/genetics , Spinal Cord/embryology , Stem Cells/metabolism
13.
Dis Model Mech ; 7(1): 107-17, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24135485

ABSTRACT

Fetal valproate syndrome (FVS) is caused by in utero exposure to the drug sodium valproate. Valproate is used worldwide for the treatment of epilepsy, as a mood stabiliser and for its pain-relieving properties. In addition to birth defects, FVS is associated with an increased risk of autism spectrum disorder (ASD), which is characterised by abnormal behaviours. Valproate perturbs multiple biochemical pathways and alters gene expression through its inhibition of histone deacetylases. Which, if any, of these mechanisms is relevant to the genesis of its behavioural side effects is unclear. Neuroanatomical changes associated with FVS have been reported and, among these, altered serotonergic neuronal differentiation is a consistent finding. Altered serotonin homeostasis is also associated with autism. Here we have used a chemical-genetics approach to investigate the underlying molecular defect in a zebrafish FVS model. Valproate causes the selective failure of zebrafish central serotonin expression. It does so by downregulating the proneural gene ascl1b, an ortholog of mammalian Ascl1, which is a known determinant of serotonergic identity in the mammalian brainstem. ascl1b is sufficient to rescue serotonin expression in valproate-treated embryos. Chemical and genetic blockade of the histone deacetylase Hdac1 downregulates ascl1b, consistent with the Hdac1-mediated silencing of ascl1b expression by valproate. Moreover, tonic Notch signalling is crucial for ascl1b repression by valproate. Concomitant blockade of Notch signalling restores ascl1b expression and serotonin expression in both valproate-exposed and hdac1 mutant embryos. Together, these data provide a molecular explanation for serotonergic defects in FVS and highlight an epigenetic mechanism for genome-environment interaction in disease.


Subject(s)
Abnormalities, Drug-Induced/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Gene Silencing , Valproic Acid/adverse effects , Zebrafish Proteins/metabolism , Abnormalities, Drug-Induced/metabolism , Animals , Anticonvulsants/chemistry , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Differentiation , Child Development Disorders, Pervasive/genetics , Disease Models, Animal , Epigenesis, Genetic , Histone Deacetylase 1/metabolism , Homeostasis , Nerve Tissue Proteins , Neurons/metabolism , Receptors, Notch/metabolism , Serotonin/metabolism , Signal Transduction , Transcription Factors , Transgenes , Valproic Acid/metabolism , Zebrafish , Zebrafish Proteins/genetics
14.
PLoS Genet ; 9(10): e1003811, 2013.
Article in English | MEDLINE | ID: mdl-24098141

ABSTRACT

Pattern formation in developing tissues is driven by the interaction of extrinsic signals with intrinsic transcriptional networks that together establish spatially and temporally restricted profiles of gene expression. How this process is orchestrated at the molecular level by genomic cis-regulatory modules is one of the central questions in developmental biology. Here we have addressed this by analysing the regulation of Pax3 expression in the context of the developing spinal cord. Pax3 is induced early during neural development in progenitors of the dorsal spinal cord and is maintained as pattern is subsequently elaborated, resulting in the segregation of the tissue into dorsal and ventral subdivisions. We used a combination of comparative genomics and transgenic assays to define and dissect several functional cis-regulatory modules associated with the Pax3 locus. We provide evidence that the coordinated activity of two modules establishes and refines Pax3 expression during neural tube development. Mutational analyses of the initiating element revealed that in addition to Wnt signaling, Nkx family homeodomain repressors restrict Pax3 transcription to the presumptive dorsal neural tube. Subsequently, a second module mediates direct positive autoregulation and feedback to maintain Pax3 expression. Together, these data indicate a mechanism by which transient external signals are converted into a sustained expression domain by the activities of distinct regulatory elements. This transcriptional logic differs from the cross-repression that is responsible for the spatiotemporal patterns of gene expression in the ventral neural tube, suggesting that a variety of circuits are deployed within the neural tube regulatory network to establish and elaborate pattern formation.


Subject(s)
Embryonic Development/genetics , Gene Regulatory Networks/genetics , Mice/genetics , Neural Tube/growth & development , Paired Box Transcription Factors/genetics , Animals , Animals, Genetically Modified , Body Patterning/genetics , Chick Embryo , DNA Mutational Analysis , DNA-Binding Proteins/genetics , Gene Expression Regulation, Developmental , Mice/metabolism , PAX3 Transcription Factor , Paired Box Transcription Factors/metabolism , Spinal Cord/growth & development , Wnt Signaling Pathway , Zebrafish/growth & development
15.
Development ; 140(8): 1740-50, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23533174

ABSTRACT

The development of a functional tissue requires coordination of the amplification of progenitors and their differentiation into specific cell types. The molecular basis for this coordination during myotome ontogeny is not well understood. Dermomytome progenitors that colonize the myotome first acquire myocyte identity and subsequently proliferate as Pax7-expressing progenitors before undergoing terminal differentiation. We show that the dynamics of sonic hedgehog (Shh) signaling is crucial for this transition in both avian and mouse embryos. Initially, Shh ligand emanating from notochord/floor plate reaches the dermomyotome, where it both maintains the proliferation of dermomyotome cells and promotes myogenic differentiation of progenitors that colonized the myotome. Interfering with Shh signaling at this stage produces small myotomes and accumulation of Pax7-expressing progenitors. An in vivo reporter of Shh activity combined with mouse genetics revealed the existence of both activator and repressor Shh activities operating on distinct subsets of cells during the epaxial myotomal maturation. In contrast to observations in mice, in avians Shh promotes the differentiation of both epaxial and hypaxial myotome domains. Subsequently, myogenic progenitors become refractory to Shh; this is likely to occur at the level of, or upstream of, smoothened signaling. The end of responsiveness to Shh coincides with, and is thus likely to enable, the transition into the growth phase of the myotome.


Subject(s)
Cell Differentiation/physiology , Gene Expression Regulation, Developmental/physiology , Hedgehog Proteins/metabolism , Muscle Development/physiology , Signal Transduction/physiology , Stem Cells/cytology , Animals , Cell Proliferation , Chick Embryo , DNA Primers/genetics , Electroporation , Genetic Vectors , Green Fluorescent Proteins/metabolism , Immunohistochemistry , In Situ Hybridization , Mice , Notochord/transplantation , PAX7 Transcription Factor/metabolism , Quail , Stem Cells/physiology , Time Factors
16.
Cell ; 148(1-2): 273-84, 2012 Jan 20.
Article in English | MEDLINE | ID: mdl-22265416

ABSTRACT

Secreted signals, known as morphogens, provide the positional information that organizes gene expression and cellular differentiation in many developing tissues. In the vertebrate neural tube, Sonic Hedgehog (Shh) acts as a morphogen to control the pattern of neuronal subtype specification. Using an in vivo reporter of Shh signaling, mouse genetics, and systems modeling, we show that a spatially and temporally changing gradient of Shh signaling is interpreted by the regulatory logic of a downstream transcriptional network. The design of the network, which links three transcription factors to Shh signaling, is responsible for differential spatial and temporal gene expression. In addition, the network renders cells insensitive to fluctuations in signaling and confers hysteresis--memory of the signal. Our findings reveal that morphogen interpretation is an emergent property of the architecture of a transcriptional network that provides robustness and reliability to tissue patterning.


Subject(s)
Gene Regulatory Networks , Hedgehog Proteins/metabolism , Neural Tube/metabolism , Signal Transduction , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Eye Proteins/genetics , Hedgehog Proteins/genetics , Homeobox Protein Nkx-2.2 , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Mice , Mice, Transgenic , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neural Stem Cells/metabolism , Oligodendrocyte Transcription Factor 2 , PAX6 Transcription Factor , Paired Box Transcription Factors/genetics , Repressor Proteins/genetics , Transcription Factors/metabolism , Zebrafish Proteins , Zinc Finger Protein Gli3
17.
Development ; 137(24): 4271-82, 2010 Dec.
Article in English | MEDLINE | ID: mdl-21098568

ABSTRACT

Sonic hedgehog signalling is essential for the embryonic development of many tissues including the central nervous system, where it controls the pattern of cellular differentiation. A genome-wide screen of neural progenitor cells to evaluate the Shh signalling-regulated transcriptome identified the forkhead transcription factor Foxj1. In both chick and mouse Foxj1 is expressed in the ventral midline of the neural tube in cells that make up the floor plate. Consistent with the role of Foxj1 in the formation of long motile cilia, floor plate cells produce cilia that are longer than the primary cilia found elsewhere in the neural tube, and forced expression of Foxj1 in neuroepithelial cells is sufficient to increase cilia length. In addition, the expression of Foxj1 in the neural tube and in an Shh-responsive cell line attenuates intracellular signalling by decreasing the activity of Gli proteins, the transcriptional mediators of Shh signalling. We show that this function of Foxj1 depends on cilia. Nevertheless, floor plate identity and ciliogenesis are unaffected in mouse embryos lacking Foxj1 and we provide evidence that additional transcription factors expressed in the floor plate share overlapping functions with Foxj1. Together, these findings identify a novel mechanism that modifies the cellular response to Shh signalling and reveal morphological and functional features of the amniote floor plate that distinguish these cells from the rest of the neuroepithelium.


Subject(s)
Cilia/metabolism , Forkhead Transcription Factors/metabolism , Hedgehog Proteins/metabolism , Neural Tube/embryology , Neural Tube/metabolism , Signal Transduction , Animals , Cells, Cultured , Chick Embryo , Chickens , Cilia/ultrastructure , Flow Cytometry , Forkhead Transcription Factors/genetics , Gene Expression Profiling , Hedgehog Proteins/genetics , Homeobox Protein Nkx-2.2 , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Immunohistochemistry , In Situ Hybridization , Mice , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , NIH 3T3 Cells , Neural Tube/ultrastructure , Transcription Factors/genetics , Transcription Factors/metabolism , Zebrafish Proteins
18.
PLoS Biol ; 8(6): e1000382, 2010 Jun 01.
Article in English | MEDLINE | ID: mdl-20532235

ABSTRACT

Morphogens are secreted signalling molecules that act in a graded manner to control the pattern of cellular differentiation in developing tissues. An example is Sonic hedgehog (Shh), which acts in several developing vertebrate tissues, including the central nervous system, to provide positional information during embryonic patterning. Here we address how Shh signalling assigns the positional identities of distinct neuronal subtype progenitors throughout the ventral neural tube. Assays of intracellular signal transduction and gene expression indicate that the duration as well as level of signalling is critical for morphogen interpretation. Progenitors of the ventral neuronal subtypes are established sequentially, with progressively more ventral identities requiring correspondingly higher levels and longer periods of Shh signalling. Moreover, cells remain sensitive to changes in Shh signalling for an extended time, reverting to antecedent identities if signalling levels fall below a threshold. Thus, the duration of signalling is important not only for the assignment but also for the refinement and maintenance of positional identity. Together the data suggest a dynamic model for ventral neural tube patterning in which positional information corresponds to the time integral of Shh signalling. This suggests an alternative to conventional models of morphogen action that rely solely on the level of signalling.


Subject(s)
Hedgehog Proteins/physiology , Neural Tube/embryology , Vertebrates/embryology , Animals , Hedgehog Proteins/metabolism , Signal Transduction
19.
Genes Dev ; 24(11): 1186-200, 2010 Jun 01.
Article in English | MEDLINE | ID: mdl-20516201

ABSTRACT

The secreted ligand Sonic Hedgehog (Shh) organizes the pattern of cellular differentiation in the ventral neural tube. For the five neuronal subtypes, increasing levels and durations of Shh signaling direct progenitors to progressively more ventral identities. Here we demonstrate that this mode of action is not applicable to the generation of the most ventral cell type, the nonneuronal floor plate (FP). In chick and mouse embryos, FP specification involves a biphasic response to Shh signaling that controls the dynamic expression of key transcription factors. During gastrulation and early somitogenesis, FP induction depends on high levels of Shh signaling. Subsequently, however, prospective FP cells become refractory to Shh signaling, and this is a prerequisite for the elaboration of their identity. This prompts a revision to the model of graded Shh signaling in the neural tube, and provides insight into how the dynamics of morphogen signaling are deployed to extend the patterning capacity of a single ligand. In addition, we provide evidence supporting a common scheme for FP specification by Shh signaling that reconciles mechanisms of FP development in teleosts and amniotes.


Subject(s)
Body Patterning/physiology , Hedgehog Proteins/metabolism , Neural Tube/cytology , Neural Tube/growth & development , Signal Transduction , Stem Cells/physiology , Animals , Biomarkers/metabolism , Chick Embryo , Down-Regulation , Embryo, Mammalian , Embryo, Nonmammalian , Female , Mice , Neurons/cytology , Somites/growth & development , Time Factors , Zebrafish
20.
Development ; 136(4): 665-76, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19168680

ABSTRACT

The progressive generation of embryonic trunk structures relies on the proper patterning of the caudal epiblast, which involves the integration of several signalling pathways. We have investigated the function of retinoic acid (RA) signalling during this process. We show that, in addition to posterior mesendoderm, primitive streak and node cells transiently express the RA-synthesizing enzyme Raldh2 prior to the headfold stage. RA-responsive cells (detected by the RA-activated RARE-lacZ transgene) are additionally found in the epiblast layer. Analysis of RA-deficient Raldh2(-/-) mutants reveals early caudal patterning defects, with an expansion of primitive streak and mesodermal markers at the expense of markers of the prospective neuroepithelium. As a result, many genes involved in neurogenesis and/or patterning of the embryonic spinal cord are affected in their expression. We demonstrate that RA signalling is required at late gastrulation stages for mesodermal and neural progenitors to respond to the Shh signal. Whole-embryo culture experiments indicate that the proper response of cells to Shh requires two RA-dependent mechanisms: (1) a balanced antagonism between Fgf and RA signals, and (2) a RA-mediated repression of Gli2 expression. Thus, an interplay between RA, Fgf and Shh signalling is likely to be an important mechanism underpinning the tight regulation of caudal embryonic development.


Subject(s)
Fibroblast Growth Factors/metabolism , Hedgehog Proteins/metabolism , Signal Transduction , Spinal Cord/embryology , Spinal Cord/metabolism , Tretinoin/metabolism , Aldehyde Oxidoreductases/deficiency , Aldehyde Oxidoreductases/genetics , Aldehyde Oxidoreductases/metabolism , Animals , Biomarkers/metabolism , Body Patterning , Embryo, Mammalian/cytology , Embryo, Mammalian/enzymology , Gastrulation , Gene Expression Regulation, Developmental , Hedgehog Proteins/genetics , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Mice , Models, Biological , Neural Plate/cytology , Neural Plate/metabolism , Neurogenesis , Primitive Streak/cytology , Primitive Streak/metabolism , Zinc Finger Protein GLI1 , Zinc Finger Protein Gli2
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