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 ProteinsABSTRACT
There is a growing interest in the mechanisms that control the apoptosis cascade during development and adult life. To investigate the regulatory events that trigger apoptosis in whole tissues, we have devised a genetically encoded caspase sensor that can be detected in live and fixed tissue by standard confocal microscopy. The sensor comprises two fluorophores, mRFP, monomeric red fluorescent protein (mRFP) and enhanced green fluorescent protein (eGFP), that are linked by an efficient and specific caspase-sensitive site. Upon caspase activation, the sensor is cleaved and eGFP translocates to the nucleus, leaving mRFP at membranes. This is detected before other markers of apoptosis, including anti-cleaved caspase 3 immunoreactivity. Moreover, the sensor does not perturb normal developmental apoptosis and is specific, as cleavage does not occur in Drosophila embryos that are unable to activate the apoptotic cascade. Importantly, dying cells can be recognized in live embryos, thus opening the way for in vivo imaging. As expected from the high conservation of caspases, it is also cleaved in dying cells of chick embryos. It is therefore likely to be generally useful to track the spatiotemporal pattern of caspase activity in a variety of species.
Subject(s)
Caspases/metabolism , Luminescent Measurements/methods , Animals , Caspases/genetics , Cell Line , Cell Survival , Chickens , Drosophila melanogaster/embryology , Drosophila melanogaster/enzymology , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/enzymology , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Spine/enzymology , Substrate Specificity , Red Fluorescent ProteinABSTRACT
Morphogens act in developing tissues to control the spatial arrangement of cellular differentiation. The activity of a morphogen has generally been viewed as a concentration-dependent response to a diffusible signal, but the duration of morphogen signalling can also affect cellular responses. One such example is the morphogen sonic hedgehog (SHH). In the vertebrate central nervous system and limbs, the pattern of cellular differentiation is controlled by both the amount and the time of SHH exposure. How these two parameters are interpreted at a cellular level has been unclear. Here we provide evidence that changing the concentration or duration of SHH has an equivalent effect on intracellular signalling. Chick neural cells convert different concentrations of SHH into time-limited periods of signal transduction, such that signal duration is proportional to SHH concentration. This depends on the gradual desensitization of cells to ongoing SHH exposure, mediated by the SHH-dependent upregulation of patched 1 (PTC1), a ligand-binding inhibitor of SHH signalling. Thus, in addition to its role in shaping the SHH gradient, PTC1 participates cell autonomously in gradient sensing. Together, the data reveal a novel strategy for morphogen interpretation, in which the temporal adaptation of cells to a morphogen integrates the concentration and duration of a signal to control differential gene expression.
Subject(s)
Hedgehog Proteins/metabolism , Signal Transduction , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Chick Embryo , Gene Expression Regulation/drug effects , Hedgehog Proteins/pharmacology , Homeobox Protein Nkx-2.2 , Homeodomain Proteins/metabolism , Mice , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neural Tube/cytology , Neural Tube/drug effects , Neural Tube/embryology , Neural Tube/metabolism , Oligodendrocyte Transcription Factor 2 , Oncogene Proteins/metabolism , PAX7 Transcription Factor/metabolism , Patched Receptors , Patched-1 Receptor , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Signal Transduction/drug effects , Time Factors , Trans-Activators/metabolism , Transcription Factors/metabolism , Zebrafish Proteins , Zinc Finger Protein GLI1ABSTRACT
During development, many signaling factors behave as morphogens, long-range signals eliciting different cellular responses according to their concentration. In ventral regions of the spinal cord, Sonic Hedgehog (Shh) is such a signal and controls the emergence, in precise spatial order, of distinct neuronal subtypes. The Gli family of transcription factors plays a central role in this process. Here we demonstrate that a gradient of Gli activity is sufficient to mediate, cell-autonomously, the full range of Shh responses in the neural tube. The incremental two- to threefold changes in Shh concentration, which determine alternative neuronal subtypes, are mimicked by similar small changes in the level of Gli activity, indicating that a gradient of Gli activity represents the intracellular correlate of graded Shh signaling. Moreover, our analysis suggests that cells integrate the level of signaling over time, consistent with the idea that signal duration, in addition to signal strength, is an important parameter controlling dorsal-ventral patterning. Together, these data indicate that Shh signaling is transduced, without amplification, into a gradient of Gli activity that orchestrates patterning of the ventral neural tube.
Subject(s)
Gene Expression Regulation, Developmental/physiology , Oncogene Proteins/metabolism , Organogenesis/physiology , Signal Transduction/physiology , Spinal Cord/embryology , Trans-Activators/metabolism , Transcription Factors/metabolism , Animals , Chick Embryo , Gene Expression Regulation, Developmental/genetics , Hedgehog Proteins , Humans , Neurons/physiology , Oncogene Proteins/genetics , Signal Transduction/genetics , Trans-Activators/genetics , Transcription Factors/genetics , Zinc Finger Protein GLI1ABSTRACT
Trunk neural crest cells are generated at the border between the neural plate and nonneural ectoderm, where they initiate a distinct program of gene expression, undergo an epithelial-mesenchymal transition (EMT), and delaminate from the neuroepithelium. Here, we provide evidence that members of three families of transcription induce these properties in premigratory neural crest cells. Sox9 acts to provide the competence for neural crest cells to undergo an EMT and is required for trunk neural crest survival. In the absence of Sox9, cells apoptose prior to or shortly after delamination. Slug/Snail, in the presence of Sox9, is sufficient to induce an EMT in neural epithelial cells, while FoxD3 regulates the expression of cell-cell adhesion molecules required for neural crest migration. Together, the data suggest a model in which a combination of transcription factors regulates the acquisition of the diverse properties of neural crest cells.
Subject(s)
Neural Crest/embryology , Animals , Apoptosis , Chick Embryo , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Embryonic Induction/genetics , Epithelium/embryology , Forkhead Transcription Factors , Gene Expression Regulation, Developmental , High Mobility Group Proteins/deficiency , High Mobility Group Proteins/genetics , High Mobility Group Proteins/physiology , Mesoderm/cytology , Mice , Mice, Knockout , Models, Biological , Repressor Proteins/genetics , Repressor Proteins/physiology , SOX9 Transcription Factor , Snail Family Transcription Factors , Transcription Factors/deficiency , Transcription Factors/genetics , Transcription Factors/physiology , Transcription, Genetic , Transfection , rhoB GTP-Binding Protein/genetics , rhoB GTP-Binding Protein/physiologyABSTRACT
An optimized assay for the binding of [3H]dimethyl-W84 to its allosteric site on M2 muscarinic receptors has been used to directly measure the affinities of allosteric ligands. Their potencies agree with those deduced indirectly by their modulation of the equilibrium binding and kinetics of [3H]N-methylscopolamine ([3H]NMS) binding to the orthosteric site. The affinities and cooperativities of orthosteric antagonists with [3H]dimethyl-W84 have also been quantitated. These affinities agree with those measured directly in a competition assay using [3H]NMS. All these data are compatible with the predictions of the allosteric ternary complex model. The association and dissociation kinetics of [3H]dimethyl-W84 are rapid but the estimate of its association rate constant is nevertheless comparable with that found for the orthosteric radioligand, [3H]NMS. This is unexpected, given that the allosteric site to which [3H]dimethyl-W84 binds is thought to be located on the external face of the receptor and above the [3H]NMS binding site that is buried within the transmembrane helices. The atypical allosteric ligands tacrine and 4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide (Duo3) inhibit [3H]dimethyl-W84 binding with the same potencies and comparably steep slope factors as found for inhibition of [3H]NMS binding. Tacrine and Duo3 decrease [3H]dimethyl-W84 affinity, not the number of binding sites. It is suggested that these atypical ligands either bind to the two known spatially separated allosteric sites on muscarinic receptors with positive cooperativity or their binding to the common allosteric site modulates receptor-receptor interactions such that homotropic positive cooperativity within a dimer or higher oligomer is generated.