RESUMO
Variability in gene expression contributes to phenotypic heterogeneity even in isogenic populations. Here, we used the stereotyped, Wnt signaling-dependent development of the Caenorhabditis elegans Q neuroblast to probe endogenous mechanisms that control gene expression variability. We found that the key Hox gene that orients Q neuroblast migration exhibits increased gene expression variability in mutants in which Wnt pathway activity has been perturbed. Distinct features of the gene expression distributions prompted us on a systematic search for regulatory interactions, revealing a network of interlocked positive and negative feedback loops. Interestingly, positive feedback appeared to cooperate with negative feedback to reduce variability while keeping the Hox gene expression at elevated levels. A minimal model correctly predicts the increased gene expression variability across mutants. Our results highlight the influence of gene network architecture on expression variability and implicate feedback regulation as an effective mechanism to ensure developmental robustness.
Assuntos
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Variação Genética , Via de Sinalização Wnt , Animais , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/genética , Movimento Celular , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Retroalimentação Fisiológica , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Redes Reguladoras de Genes , Glicoproteínas/genética , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Proteínas WntRESUMO
How the interplay between cell- and tissue-level processes produces correctly proportioned organs is a key problem in biology. In plants, the relative size of leaves compared with their lateral appendages, called stipules, varies tremendously throughout development and evolution, yet relevant mechanisms remain unknown. Here we use genetics, live imaging, and modeling to show that in Arabidopsis leaves, the LATE MERISTEM IDENTITY1 (LMI1) homeodomain protein regulates stipule proportions via an endoreduplication-dependent trade-off that limits tissue size despite increasing cell growth. LM1 acts through directly activating the conserved mitosis blocker WEE1, which is sufficient to bypass the LMI1 requirement for leaf proportionality.
Assuntos
Proteínas de Arabidopsis/fisiologia , Endorreduplicação , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/fisiologia , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Folhas de Planta/anatomia & histologia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Here we investigate mechanisms underlying the diversification of biological forms using crucifer leaf shape as an example. We show that evolution of an enhancer element in the homeobox gene REDUCED COMPLEXITY (RCO) altered leaf shape by changing gene expression from the distal leaf blade to its base. A single amino acid substitution evolved together with this regulatory change, which reduced RCO protein stability, preventing pleiotropic effects caused by its altered gene expression. We detected hallmarks of positive selection in these evolved regulatory and coding sequence variants and showed that modulating RCO activity can improve plant physiological performance. Therefore, interplay between enhancer and coding sequence evolution created a potentially adaptive path for morphological evolution.
Assuntos
Arabidopsis/fisiologia , Cardamine/anatomia & histologia , Cardamine/genética , Evolução Molecular , Folhas de Planta/anatomia & histologia , Folhas de Planta/genética , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Cardamine/classificação , Elementos Facilitadores Genéticos/genética , Perfilação da Expressão Gênica , Genes de Plantas/genéticaRESUMO
Van Gogh-like (Vangl) and Prickle (Pk) are core components of the non-canonical Wnt planar cell polarity pathway that controls epithelial polarity and cell migration. Studies in vertebrate model systems have suggested that Vangl and Pk may also inhibit signaling through the canonical Wnt/ß-catenin pathway, but the functional significance of this potential cross-talk is unclear. In the nematode C. elegans, the Q neuroblasts and their descendants migrate in opposite directions along the anteroposterior body axis. The direction of these migrations is specified by Wnt signaling, with activation of canonical Wnt signaling driving posterior migration, and non-canonical Wnt signaling anterior migration. Here, we show that the Vangl ortholog VANG-1 influences the Wnt signaling response of the Q neuroblasts by negatively regulating canonical Wnt signaling. This inhibitory activity depends on a carboxy-terminal PDZ binding motif in VANG-1 and the Dishevelled ortholog MIG-5, but is independent of the Pk ortholog PRKL-1. Moreover, using Vangl1 and Vangl2 double mutant cells, we show that a similar mechanism acts in mammalian cells. We conclude that cross-talk between VANG-1/Vangl and the canonical Wnt pathway is an evolutionarily conserved mechanism that ensures robust specification of Wnt signaling responses.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Fosfoproteínas/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Padronização Corporal/fisiologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Linhagem da Célula , Polaridade Celular/genética , Polaridade Celular/fisiologia , Proteínas Desgrenhadas/genética , Proteínas Desgrenhadas/metabolismo , Genes de Helmintos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Fosfoproteínas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Regulation of gene expression by signaling pathways often occurs through a transcriptional switch, where the transcription factor responsible for signal-dependent gene activation represses the same targets in the absence of signaling. T-cell factors (TCFs) are transcription factors in the Wnt/ß-catenin pathway, which control numerous cell fate specification events in metazoans. The TCF transcriptional switch is mediated by many co-regulators that contribute to repression or activation of Wnt target genes. It is typically assumed that DNA recognition by TCFs is important for target gene location, but plays no role in the actual switch. TCF/Pangolin (the fly TCF) and some vertebrate TCF isoforms bind DNA through two distinct domains, a High Mobility Group (HMG) domain and a C-clamp, which recognize DNA motifs known as HMG and Helper sites, respectively. Here, we demonstrate that POP-1 (the C. elegans TCF) also activates target genes through HMG and Helper site interactions. Helper sites enhanced the ability of a synthetic enhancer to detect Wnt/ß-catenin signaling in several tissues and revealed an unsuspected role for POP-1 in regulating the C. elegans defecation cycle. Searching for HMG-Helper site clusters allowed the identification of a new POP-1 target gene active in the head muscles and gut. While Helper sites and the C-clamp are essential for activation of worm and fly Wnt targets, they are dispensable for TCF-dependent repression of targets in the absence of Wnt signaling. These data suggest that a fundamental change in TCF-DNA binding contributes to the transcriptional switch that occurs upon Wnt stimulation.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Regulação da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas Repressoras/metabolismo , Animais , Sítios de Ligação , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Domínios HMG-Box/genética , Proteínas de Grupo de Alta Mobilidade/genética , Motivos de Nucleotídeos/genética , Ligação Proteica , Proteínas Repressoras/genética , Transdução de Sinais/genética , Via de Sinalização Wnt/genéticaRESUMO
Establishment of cell polarity is crucial for many biological processes including cell migration and asymmetric cell division. The establishment of cell polarity consists of two sequential processes: an external gradient is first sensed and then the resulting signal is amplified and maintained by intracellular signaling networks usually using positive feedback regulation. Generally, these two processes are intertwined and it is challenging to determine which proteins contribute to the sensing or amplification process, particularly in multicellular organisms. Here, we integrated phenomenological modeling with quantitative single-cell measurements to separate the sensing and amplification components of Wnt ligands and receptors during establishment of polarity of the Caenorhabditis elegans P cells. By systematically exploring how P-cell polarity is altered in Wnt ligand and receptor mutants, we inferred that ligands predominantly affect the sensing process, whereas receptors are needed for both sensing and amplification. This integrated approach is generally applicable to other systems and will facilitate decoupling of the different layers of signal sensing and amplification.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Polaridade Celular/fisiologia , Receptores Wnt/metabolismo , Proteínas Wnt/metabolismo , Animais , Divisão Celular Assimétrica , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Mutação , Receptores Wnt/genética , Análise de Célula Única , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Wnt/genética , beta Catenina/metabolismoRESUMO
A key challenge in biology is to understand how the regional control of cell growth gives rise to final organ forms. Plant leaves must coordinate growth along both the proximodistal and mediolateral axes to produce their final shape. However, the cell-level mechanisms controlling this coordination remain largely unclear. Here, we show that, in A. thaliana, WOX5, one of the WUSCHEL-RELATED HOMEOBOX (WOX) family of homeobox genes, acts redundantly with WOX1 and WOX3 (PRESSED FLOWER [PRS]) to control leaf shape. Through genetics and hormone measurements, we find that these WOXs act in part through the regional control of YUCCA (YUC) auxin biosynthetic gene expression along the leaf margin. The requirement for WOX-mediated YUC expression in patterning of leaf shape cannot be bypassed by the epidermal expression of YUC, indicating that the precise domain of auxin biosynthesis is important for leaf form. Using time-lapse growth analysis, we demonstrate that WOX-mediated auxin biosynthesis organizes a proximodistal growth gradient that promotes lateral growth and consequently the characteristic ellipsoid A. thaliana leaf shape. We also provide evidence that WOX proteins shape the proximodistal gradient of differentiation by inhibiting differentiation proximally in the leaf blade and promoting it distally. This regulation allows sustained growth of the blade and enables a leaf to attain its final form. In conclusion, we show that the WOX/auxin regulatory module shapes leaf form by coordinating growth along the proximodistal and mediolateral leaf axes.
Assuntos
Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Organogênese Vegetal/genética , Folhas de Planta/crescimento & desenvolvimento , Arabidopsis/anatomia & histologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Ácidos Indolacéticos/metabolismo , Microscopia Intravital , Oxigenases/genética , Oxigenases/metabolismo , Folhas de Planta/anatomia & histologia , Plantas Geneticamente Modificadas , Imagem com Lapso de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The origin of a terrestrial flora in the Ordovician required adaptation to novel biotic and abiotic stressors. Oil bodies, a synapomorphy of liverworts, accumulate secondary metabolites, but their function and development are poorly understood. Oil bodies of Marchantia polymorpha develop within specialized cells as one single large organelle. Here, we show that a class I homeodomain leucine-zipper (C1HDZ) transcription factor controls the differentiation of oil body cells in two different ecotypes of the liverwort M. polymorpha, a model genetic system for early divergent land plants. In flowering plants, these transcription factors primarily modulate responses to abiotic stress, including drought. However, loss-of-function alleles of the single ortholog gene, MpC1HDZ, in M. polymorpha did not exhibit phenotypes associated with abiotic stress. Rather, Mpc1hdz mutant plants were more susceptible to herbivory, and total plant extracts of the mutant exhibited reduced antibacterial activity. Transcriptomic analysis of the mutant revealed a reduction in expression of genes related to secondary metabolism that was accompanied by a specific depletion of oil body terpenoid compounds. Through time-lapse imaging, we observed that MpC1HDZ expression maxima precede oil body formation, indicating that MpC1HDZ mediates differentiation of oil body cells. Our results indicate that M. polymorpha oil bodies, and MpC1HDZ, are critical for defense against herbivory, but not for abiotic stress tolerance. Thus, C1HDZ genes were co-opted to regulate separate responses to biotic and abiotic stressors in two distinct land plant lineages.
Assuntos
Proteínas de Arabidopsis/fisiologia , Artrópodes , Herbivoria , Gotículas Lipídicas/metabolismo , Marchantia/genética , Marchantia/metabolismo , Proteínas Mitocondriais/fisiologia , Transportadores de Ácidos Monocarboxílicos/fisiologia , Óleos de Plantas/metabolismo , Fenômenos Fisiológicos Vegetais/genética , Animais , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Expressão Gênica , Zíper de Leucina/fisiologia , Marchantia/fisiologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
Mechanisms through which the evolution of gene regulation causes morphological diversity are largely unclear. The tremendous shape variation among plant leaves offers attractive opportunities to address this question. In cruciferous plants, the REDUCED COMPLEXITY (RCO) homeodomain protein evolved via gene duplication and acquired a novel expression domain that contributed to leaf shape diversity. However, the molecular pathways through which RCO regulates leaf growth are unknown. A key question is to identify genome-wide transcriptional targets of RCO and the DNA sequences to which RCO binds. We investigate this question using Cardamine hirsuta, which has complex leaves, and its relative Arabidopsis thaliana, which evolved simple leaves through loss of RCO. We demonstrate that RCO directly regulates genes controlling homeostasis of the hormone cytokinin to repress growth at the leaf base. Elevating cytokinin signaling in the RCO expression domain is sufficient to both transform A. thaliana simple leaves into complex ones and partially bypass the requirement for RCO in C. hirsuta complex leaf development. We also identify RCO as its own target gene. RCO directly represses its own transcription via an array of low-affinity binding sites, which evolved after RCO duplicated from its progenitor sequence. This autorepression is required to limit RCO expression. Thus, evolution of low-affinity binding sites created a negative autoregulatory loop that facilitated leaf shape evolution by defining RCO expression and fine-tuning cytokinin activity. In summary, we identify a transcriptional mechanism through which conflicts between novelty and pleiotropy are resolved during evolution and lead to morphological differences between species.
Assuntos
Citocininas/metabolismo , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cardamine/genética , Cardamine/metabolismo , Citocininas/genética , Evolução Molecular , Duplicação Gênica/genética , Regulação da Expressão Gênica de Plantas/genética , Homeostase , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
An open problem in biology is to derive general principles that capture how morphogenesis evolved to generate diverse forms in different organisms. Here we discuss recent work investigating the morphogenetic basis for digit loss in vertebrate limbs and variation in form of marginal outgrowths of angiosperm (flowering plant) leaves. Two pathways underlie digit loss in vertebrate limbs. First, alterations to digit patterning arise through modification of expression of the Patched 1 receptor, which senses the Sonic Hedgehog morphogen and limits its mobility in the limb bud. Second, evolutionary changes to the degree of programmed cell death between digits influence their development after their initiation. Similarly, evolutionary modification of leaf margin outgrowths occurs via two broad pathways. First, species-specific transcription factor expression modulates outgrowth patterning dependent on regulated transport of the hormone auxin. Second, species-specific expression of the newly discovered REDUCED COMPLEXITY homeodomain transcription factor influences growth between individual outgrowths after their initiation. These findings demonstrate that in both plants and animals tinkering with either patterning or post-patterning processes can cause morphological change. They also highlight the considerable flexibility of morphological evolution and indicate that it may be possible to derive broad principles that capture how morphogenesis evolved across complex eukaryotes.
RESUMO
Members of the Wnt family of secreted signaling proteins are key regulators of cell migration and axon guidance. In the nematode C. elegans, the migration of the QR neuroblast descendants requires multiple Wnt ligands and receptors. We found that the migration of the QR descendants is divided into three sequential phases that are each mediated by a distinct Wnt signaling mechanism. Importantly, the transition from the first to the second phase, which is the main determinant of the final position of the QR descendants along the anteroposterior body axis, is mediated through a cell-autonomous process in which the time-dependent expression of a Wnt receptor turns on the canonical Wnt/ß-catenin signaling response that is required to terminate long-range anterior migration. Our results show that, in addition to direct guidance of cell migration by Wnt morphogenic gradients, cell migration can also be controlled indirectly through cell-intrinsic modulation of Wnt signaling responses.
Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Movimento Celular/genética , Células-Tronco Neurais/fisiologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/genética , Animais , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/biossíntese , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Polaridade Celular , Receptores Frizzled/biossíntese , Receptores Frizzled/metabolismo , Regulação da Expressão Gênica/genética , Glicoproteínas/biossíntese , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Morfogênese , Células-Tronco Neurais/citologia , Fosfoproteínas/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Receptores Acoplados a Proteínas G/biossíntese , Fatores de Transcrição/genética , Proteínas Wnt/biossíntese , beta Catenina/metabolismoRESUMO
Most secreted proteins in eukaryotes are modified on the amino acid consensus sequence NxS/T by an N-glycan through the process of N-glycosylation. The N-glycans on glycoproteins are processed in the endoplasmic reticulum (ER) to different mannose-type N-glycans or, when the protein passes through the Golgi apparatus, to different complex glycan forms. Here we describe the capturing of N-glycopeptides from a trypsin digest of total protein extracts of Arabidopsis plants and release of these captured peptides following Peptide N-glycosidase (PNGase) treatment for analysis of N-glycan site-occupancy. The mixture of peptides released as a consequence of the PNGase treatment was analyzed by two dimensional nano-LC-MS. As the PNGase treatment of glycopeptides results in the deamidation of the asparagine (N) in the NxS/T site of the released peptide, this asparagine (N) to aspartic acid (D) conversion is used as a glycosylation 'signature'. The efficiency of PNGase F and PNGase A in peptide release is discussed. The identification of proteins with a single glycopeptide was limited by the used search algorithm but could be improved using a reference database including deamidated peptide sequences. Additional stringency settings were used for filtering results to minimize false discovery. This resulted in identification of 330 glycopeptides on 173 glycoproteins from Arabidopsis, of which 28 putative glycoproteins, that were previously not annotated as secreted protein in The Arabidopsis Information Resource database (TAIR). Furthermore, the identified glycosylation site occupancy helped to determine the correct topology for membrane proteins. A quantitative comparison of peptide signal was made between wild type and complex-glycan-less (cgl) mutant Arabidopsis from three replicate leaf samples using a label-free MS peak comparison. As an example, the identified membrane protein SKU5 (AT4G12420) showed differential glycopeptide intensity ratios between WT and cgl indicating heterogeneous glycan modification on single protein. BIOLOGICAL SIGNIFICANCE: Proteins that enter the secretory pathway are mostly modified by N-glycans. The function of N-glycosylation has been well studied in mammals. However, in plants the function of N-glycosylation is still unclear, because glycosylation mutants in plants often do not have a clear phenotype. Here we analyzed which proteins are modified by N-glycans in plants by developing a glycopeptide enrichment method for plant proteins. Subsequently, label free comparative proteomics was employed using protein fractions from wild type and from a mutant which is blocked in modification of the N-glycan into complex glycans. The results provide new information on N-glycosylation sites on numerous secreted proteins. Results allow for specific mapping of multiple glycosylation site occupancy on proteins, which provides information on which glycosylation sites are protected or non-used from downstream processing and thus presumably are buried into the protein structure. Glycoproteomics can therefore contribute to protein structure analysis. Indeed, mapping the glycosylation sites on membrane proteins gives information on the topology of protein folds over the membrane. We thus were able to correct the topology prediction of three membrane proteins. Besides, these studies also identified limitations in the software that is used to identify single modified peptide per protein. This article is part of a Special Issue entitled: Translational Plant Proteomics.
Assuntos
Arabidopsis/química , Glicoproteínas/química , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Glicopeptídeos/isolamento & purificação , Glicosilação , Glicoproteínas de Membrana/química , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismoRESUMO
In eukaryotes, proteins that are secreted into the ER are mostly modified by N-glycans on consensus NxS/T sites. The N-linked glycan subsequently undergoes varying degrees of processing by enzymes which are spatially distributed over the ER and the Golgi apparatus. The post-ER N-glycan processing to complex glycans differs between animals and plants, with consequences for N-glycan and glycopeptide isolation and characterization of plant glycoproteins. Here we describe some recent developments in plant glycoproteomics and illustrate how general and plant specific technologies may be used to address different important biological questions.