RESUMEN
During development, morphogens pattern tissues by instructing cell fate across long distances. Directly visualizing morphogen transport in situ has been inaccessible, so the molecular mechanisms ensuring successful morphogen delivery remain unclear. To tackle this longstanding problem, we developed a mouse model for compromised sonic hedgehog (SHH) morphogen delivery and discovered that endocytic recycling promotes SHH loading into signaling filopodia called cytonemes. We optimized methods to preserve in vivo cytonemes for advanced microscopy and show endogenous SHH localized to cytonemes in developing mouse neural tubes. Depletion of SHH from neural tube cytonemes alters neuronal cell fates and compromises neurodevelopment. Mutation of the filopodial motor myosin 10 (MYO10) reduces cytoneme length and density, which corrupts neuronal signaling activity of both SHH and WNT. Combined, these results demonstrate that cytoneme-based signal transport provides essential contributions to morphogen dispersion during mammalian tissue development and suggest MYO10 is a key regulator of cytoneme function.
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Estructuras de la Membrana Celular , Miosinas , Tubo Neural , Transducción de Señal , Animales , Ratones , Transporte Biológico , Estructuras de la Membrana Celular/metabolismo , Proteínas Hedgehog/metabolismo , Miosinas/metabolismo , Seudópodos/metabolismo , Tubo Neural/citología , Tubo Neural/metabolismoRESUMEN
Intercellular signaling molecules, known as morphogens, act at a long range in developing tissues to provide spatial information and control properties such as cell fate and tissue growth. The production, transport, and removal of morphogens shape their concentration profiles in time and space. Downstream signaling cascades and gene regulatory networks within cells then convert the spatiotemporal morphogen profiles into distinct cellular responses. Current challenges are to understand the diverse molecular and cellular mechanisms underlying morphogen gradient formation, as well as the logic of downstream regulatory circuits involved in morphogen interpretation. This knowledge, combining experimental and theoretical results, is essential to understand emerging properties of morphogen-controlled systems, such as robustness and scaling.
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During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here, we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a "trimer-of-trimers" super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs.
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Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas Ligadas a GPI/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Oncogénicas/metabolismo , Animales , Moléculas de Adhesión Celular Neuronal/química , Movimiento Celular , Receptor DCC/deficiencia , Receptor DCC/genética , Proteínas Ligadas a GPI/química , Conos de Crecimiento/fisiología , Humanos , Ventrículos Laterales/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/química , Neuronas/citología , Neuronas/metabolismo , Proteínas Oncogénicas/química , Proteínas Oncogénicas/genética , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de SeñalRESUMEN
Communication between cells pervades the development and physiology of metazoans. In animals, this process is carried out by a relatively small number of signaling pathways, each consisting of a chain of biochemical events through which extracellular stimuli control the behavior of target cells. One such signaling system is the Hedgehog pathway, which is crucial in embryogenesis and is implicated in many birth defects and cancers. Although Hedgehog pathway components were identified by genetic analysis more than a decade ago, our understanding of the molecular mechanisms of signaling is far from complete. In this review, we focus on the biochemistry and cell biology of the Hedgehog pathway. We examine the unique biosynthesis of the Hedgehog ligand, its specialized release from cells into extracellular space, and the poorly understood mechanisms involved in ligand reception and pathway activation at the surface of target cells. We highlight several critical questions that remain open.
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Proteínas Hedgehog/metabolismo , Transducción de Señal , Animales , Retroalimentación Fisiológica , Humanos , Ligandos , Modelos BiológicosRESUMEN
Axon guidance relies on a combinatorial code of receptor and ligand interactions that direct adhesive/attractive and repulsive cellular responses. Recent structural data have revealed many of the molecular mechanisms that govern these interactions and enabled the design of sophisticated mutant tools to dissect their biological functions. Here, we discuss the structure/function relationships of four major classes of guidance cues (ephrins, semaphorins, slits, netrins) and examples of morphogens (Wnt, Shh) and of cell adhesion molecules (FLRT). These cell signaling systems rely on specific modes of receptor-ligand binding that are determined by selective binding sites; however, defined structure-encoded receptor promiscuity also enables cross talk between different receptor/ligand families and can also involve extracellular matrix components. A picture emerges in which a multitude of highly context-dependent structural assemblies determines the finely tuned cellular behavior required for nervous system development.
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Orientación del Axón , Proteínas del Tejido Nervioso/metabolismo , Animales , Humanos , Modelos Biológicos , Receptores de Superficie Celular/metabolismo , Transducción de SeñalRESUMEN
Graded transcription factors are pivotal regulators of embryonic patterning, but whether their role changes over time is unclear. A light-regulated protein degradation system was used to assay temporal dependence of the transcription factor Dorsal in dorsal-ventral axis patterning of Drosophila embryos. Surprisingly, the high-threshold target gene snail only requires Dorsal input early but not late when Dorsal levels peak. Instead, late snail expression can be supported by action of the Twist transcription factor, specifically, through one enhancer, sna.distal This study demonstrates that continuous input is not required for some Dorsal targets and downstream responses, such as twist, function as molecular ratchets.
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Tipificación del Cuerpo/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Factores de Transcripción/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , Animales , Tipificación del Cuerpo/efectos de la radiación , Proteínas de Drosophila/genética , Embrión no Mamífero , Luz , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteolisis/efectos de la radiación , Factores de Transcripción de la Familia Snail/metabolismo , Factores de Transcripción/genética , Proteína 1 Relacionada con Twist/genéticaRESUMEN
Heterocyst differentiation that occurs in some filamentous cyanobacteria, such as Anabaena sp. PCC 7120, provides a unique model for prokaryotic developmental biology. Heterocyst cells are formed in response to combined-nitrogen deprivation and possess a microoxic environment suitable for nitrogen fixation following extensive morphological and physiological reorganization. A filament of Anabaena is a true multicellular organism, as nitrogen and carbon sources are exchanged among different cells and cell types through septal junctions to ensure filament growth. Because heterocysts are terminally differentiated cells and unable to divide, their activity is an altruistic behavior dedicated to providing fixed nitrogen for neighboring vegetative cells. Heterocyst development is also a process of one-dimensional pattern formation, as heterocysts are semiregularly intercalated among vegetative cells. Morphogens form gradients along the filament and interact with each other in a fashion that fits well into the Turing model, a mathematical framework to explain biological pattern formation.
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Anabaena , Cianobacterias , Anabaena/metabolismo , Proteínas Bacterianas/metabolismo , Cianobacterias/metabolismo , Regulación Bacteriana de la Expresión Génica , Nitrógeno/metabolismo , Fijación del NitrógenoRESUMEN
Cortical arealization arises during neurodevelopment from the confluence of molecular gradients representing patterned expression of morphogens and transcription factors. However, whether similar gradients are maintained in the adult brain remains unknown. Here, we uncover three axes of topographic variation in gene expression in the adult human brain that specifically capture previously identified rostral-caudal, dorsal-ventral, and medial-lateral axes of early developmental patterning. The interaction of these spatiomolecular gradients i) accurately reconstructs the position of brain tissue samples, ii) delineates known functional territories, and iii) can model the topographical variation of diverse cortical features. The spatiomolecular gradients are distinct from canonical cortical axes differentiating the primary sensory cortex from the association cortex, but radiate in parallel with the axes traversed by local field potentials along the cortex. We replicate all three molecular gradients in three independent human datasets as well as two nonhuman primate datasets and find that each gradient shows a distinct developmental trajectory across the lifespan. The gradients are composed of several well-known transcription factors (e.g., PAX6 and SIX3), and a small set of genes shared across gradients are strongly enriched for multiple diseases. Together, these results provide insight into the developmental sculpting of functionally distinct brain regions, governed by three robust transcriptomic axes embedded within brain parenchyma.
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Encéfalo , Humanos , Encéfalo/metabolismo , Animales , Adulto , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Factor de Transcripción PAX6/metabolismo , Factor de Transcripción PAX6/genética , Regulación del Desarrollo de la Expresión Génica , Masculino , Tipificación del Cuerpo/genética , Femenino , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genéticaRESUMEN
Morphogen gradients impart positional information to cells in a homogenous tissue field. Fgf8a, a highly conserved growth factor, has been proposed to act as a morphogen during zebrafish gastrulation. However, technical limitations have so far prevented direct visualization of the endogenous Fgf8a gradient and confirmation of its morphogenic activity. Here, we monitor Fgf8a propagation in the developing neural plate using a CRISPR/Cas9-mediated EGFP knock-in at the endogenous fgf8a locus. By combining sensitive imaging with single-molecule fluorescence correlation spectroscopy, we demonstrate that Fgf8a, which is produced at the embryonic margin, propagates by diffusion through the extracellular space and forms a graded distribution towards the animal pole. Overlaying the Fgf8a gradient curve with expression profiles of its downstream targets determines the precise input-output relationship of Fgf8a-mediated patterning. Manipulation of the extracellular Fgf8a levels alters the signaling outcome, thus establishing Fgf8a as a bona fide morphogen during zebrafish gastrulation. Furthermore, by hindering Fgf8a diffusion, we demonstrate that extracellular diffusion of the protein from the source is crucial for it to achieve its morphogenic potential.
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Factores de Crecimiento de Fibroblastos , Gastrulación , Proteínas de Pez Cebra , Pez Cebra , Animales , Tipificación del Cuerpo/genética , Gastrulación/genética , Morfogénesis/genética , Transducción de Señal/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismoRESUMEN
Tissue patterning during embryonic development is remarkably precise. Here, we numerically determine the impact of the cell diameter, gradient length and the morphogen source on the variability of morphogen gradients. We show that the positional error increases with the gradient length relative to the size of the morphogen source, and with the square root of the cell diameter and the readout position. We provide theoretical explanations for these relationships, and show that they enable high patterning precision over developmental time for readouts that scale with expanding tissue domains, as observed in the Drosophila wing disc. Our analysis suggests that epithelial tissues generally achieve higher patterning precision with small cross-sectional cell areas. An extensive survey of measured apical cell areas shows that they are indeed small in developing tissues that are patterned by morphogen gradients. Enhanced precision may thus have led to the emergence of pseudostratification in epithelia, a phenomenon for which the evolutionary benefit had so far remained elusive.
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Proteínas de Drosophila , Drosophila , Animales , Estudios Transversales , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Desarrollo Embrionario , Tamaño de la Célula , Tipificación del Cuerpo/genética , Regulación del Desarrollo de la Expresión Génica , Modelos Biológicos , Drosophila melanogaster/genéticaRESUMEN
Receptor tyrosine kinases exhibit ligand-induced activity and uptake into cells via endocytosis. In the case of epidermal growth factor (EGF) receptor (EGFR), the resulting endosomes are trafficked to the perinuclear region, where dephosphorylation of receptors occurs, which are subsequently directed to degradation. Traveling endosomes bearing phosphorylated EGFRs are subjected to the activity of cytoplasmic phosphatases as well as interactions with the endoplasmic reticulum (ER). The peri-nuclear region harbors ER-embedded phosphatases, a component of the EGFR-bearing endosome-ER contact site. The ER is also emerging as a central player in spatiotemporal control of endosomal motility, positioning, tubulation, and fission. Past studies strongly suggest that the physical interaction between the ER and endosomes forms a reaction "unit" for EGFR dephosphorylation. Independently, endosomes have been implicated to enable quantization of EGFR signals by modulation of the phosphorylation levels. Here, we review the distinct mechanisms by which endosomes form the logistical means for signal quantization and speculate on the role of the ER.
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Orientational order, encoded in anisotropic fields, plays an important role during the development of an organism. A striking example of this is the freshwater polyp Hydra, where topological defects in the muscle fiber orientation have been shown to localize to key features of the body plan. This body plan is organized by morphogen concentration gradients, raising the question how muscle fiber orientation, morphogen gradients and body shape interact. Here, we introduce a minimal model that couples nematic orientational order to the gradient of a morphogen field. We show that on a planar surface, alignment to a radial concentration gradient can induce unbinding of topological defects, as observed during budding and tentacle formation in Hydra, and stabilize aster/vortex-like defects, as observed at a Hydra's mouth. On curved surfaces mimicking the morphologies of Hydra in various stages of development-from spheroid to adult-our model reproduces the experimentally observed reorganization of orientational order. Our results suggest how gradient alignment and curvature effects may work together to control orientational order during development and lay the foundations for future modeling efforts that will include the tissue mechanics that drive shape deformations.
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Hydra , Animales , Anisotropía , Morfogénesis , Hydra/fisiología , Regeneración/fisiología , Tipificación del CuerpoRESUMEN
Cancer-associated fibroblasts (CAFs) are a crucial component in the tumor microenvironment influencing cancer progression. Besides shaping the extracellular matrix, these fibroblasts provide signaling factors to facilitate tumor survival and alter tumor behavior. In gastric cancer, one crucial signaling pathway influencing invasion and metastasis is the Wnt/Planar Cell Polarity (PCP) signaling. The crucial PCP ligand in this context is WNT5A, which is produced by the CAFs, and gastric cancer cells react upon this signal by enhanced polarized migration. Why gastric cancer cells respond to this signal is still unclear, as their expression level for the central WNT5A receptor, ROR2, is very low. Here, we show that CAFs display long and branched filopodia that form an extensive, complex network engulfing gastric cancer cells, such as the gastric cancer cell line AGS. CAFs have a significantly higher expression level of ROR2 than normal gastric fibroblasts and AGS cells. By high-resolution imaging, we observe a direct transfer of fluorescently tagged ROR2 from CAF to AGS cells by signaling filopodia, known as cytonemes. Surprisingly, we find that the transferred ROR2 complexes can activate Wnt/JNK signaling in AGS cells. Consistently, blockage of ROR2 function in the CAFs leads to reduced paracrine Wnt/JNK signaling, cell polarization, and migration of the receiving AGS cells. Complementary, enhanced migration via paracrine ROR2 transfer was observed in a zebrafish in vivo model. These findings demonstrate a fresh role for cytoneme-mediated signaling in the tumor microenvironment. Cytonemes convey Wnt receptors from CAFs to gastric cancer cells, allowing them to respond to Wnt/PCP signals.
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Fibroblastos Asociados al Cáncer , Neoplasias Gástricas , Animales , Neoplasias Gástricas/genética , Microambiente Tumoral , Vía de Señalización Wnt , Pez Cebra , Humanos , Línea Celular TumoralRESUMEN
Heparan sulfate proteoglycans (HSPGs) are composed of a core protein and glycosaminoglycan (GAG) chains and serve as coreceptors for many growth factors and morphogens. To understand the molecular mechanisms by which HSPGs regulate morphogen gradient formation and signaling, it is important to determine the relative contributions of the carbohydrate and protein moieties to the proteoglycan function. To address this question, we generated ΔGAG alleles for dally and dally-like protein (dlp), two Drosophila HSPGs of the glypican family, in which all GAG-attachment serine residues are substituted to alanine residues using CRISPR/Cas9 mutagenesis. In these alleles, the glypican core proteins are expressed from the endogenous loci with no GAG modification. Analyses of the dallyΔGAG allele defined Dally functions that do not require heparan sulfate (HS) chains and that need both core protein and HS chains. We found a new, dallyΔGAG-specific phenotype, the formation of a posterior ectopic vein, which we have never seen in the null mutants. Unlike dallyΔGAG, dlpΔGAG mutants do not show most of the dlp null mutant phenotypes, suggesting that HS chains are dispensable for these dlp functions. As an exception, HS is essentially required for Dlp's activity at the neuromuscular junction. Thus, Drosophila glypicans show strikingly different levels of HS dependency. The ΔGAG mutant alleles of the glypicans serve as new molecular genetic toolsets highly useful to address important biological questions, such as molecular mechanisms of morphogen gradient formation.
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Proteínas de Drosophila , Drosophila melanogaster , Glipicanos , Heparitina Sulfato , Animales , Proteínas de Drosophila/metabolismo , Glipicanos/genética , Glipicanos/química , Glipicanos/metabolismo , Proteoglicanos de Heparán Sulfato/genética , Proteoglicanos de Heparán Sulfato/metabolismo , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Glicoproteínas de Membrana/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismoRESUMEN
Morphogens provide quantitative and robust signaling systems to achieve stereotypic patterning and morphogenesis. Heparan sulfate (HS) proteoglycans (HSPGs) are key components of such regulatory feedback networks. In Drosophila, HSPGs serve as co-receptors for a number of morphogens, including Hedgehog (Hh), Wingless (Wg), Decapentaplegic (Dpp) and Unpaired (Upd, or Upd1). Recently, Windpipe (Wdp), a chondroitin sulfate (CS) proteoglycan (CSPG), was found to negatively regulate Upd and Hh signaling. However, the roles of Wdp, and CSPGs in general, in morphogen signaling networks are poorly understood. We found that Wdp is a major CSPG with 4-O-sulfated CS in Drosophila. Overexpression of wdp modulates Dpp and Wg signaling, showing that it is a general regulator of HS-dependent pathways. Although wdp mutant phenotypes are mild in the presence of morphogen signaling buffering systems, this mutant in the absence of Sulf1 or Dally, molecular hubs of the feedback networks, produces high levels of synthetic lethality and various severe morphological phenotypes. Our study indicates a close functional relationship between HS and CS, and identifies the CSPG Wdp as a novel component in morphogen feedback pathways.
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Proteínas de Drosophila , Drosophila , Animales , Proteoglicanos Tipo Condroitín Sulfato/genética , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteoglicanos de Heparán Sulfato/genética , Proteoglicanos de Heparán Sulfato/metabolismo , Sulfatasas/genética , Sulfatasas/metabolismo , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMEN
The Turing model (or reaction-diffusion model), first published in 1952, is a mathematical model that can account for autonomy in the morphogenesis of organisms. Although initially controversial, the model has gradually gained wider acceptance among experimental embryologists due to the accumulation of experimental data to support it. More recently, this model and others based on it have been used not only to explain biological phenomena conceptually but also as working hypotheses for molecular-level experiments and as internal components of more-complex 3D models. In this Spotlight, I will provide a personal perspective from an experimental biologist on some of the recent developments of the Turing model.
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Tipificación del Cuerpo , Modelos Biológicos , Morfogénesis , Difusión , Biología EvolutivaRESUMEN
In many developing and regenerating systems, tissue pattern is established through gradients of informative morphogens, but we know little about how cells interpret these. Using experimental manipulation of early chick embryos, including misexpression of an inducer (VG1 or ACTIVIN) and an inhibitor (BMP4), we test two alternative models for their ability to explain how the site of primitive streak formation is positioned relative to the rest of the embryo. In one model, cells read morphogen concentrations cell-autonomously. In the other, cells sense changes in morphogen status relative to their neighbourhood. We find that only the latter model can account for the experimental results, including some counter-intuitive predictions. This mechanism (which we name the 'neighbourhood watch' model) illuminates the classic 'French Flag Problem' and how positional information is interpreted by a sheet of cells in a large developing system.
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Gastrulación , Estratos Germinativos , Animales , Embrión de Pollo , GástrulaRESUMEN
Transcription in the early Drosophila blastoderm is coordinated by the collective action of hundreds of enhancers. Many genes are controlled by so-called 'shadow enhancers', which provide resilience to environment or genetic insult, allowing the embryo to robustly generate a precise transcriptional pattern. Emerging evidence suggests that many shadow enhancer pairs do not drive identical expression patterns, but the biological significance of this remains unclear. In this study, we characterize the shadow enhancer pair controlling the gene short gastrulation (sog). We removed either the intronic proximal enhancer or the upstream distal enhancer and monitored sog transcriptional kinetics. Notably, each enhancer differs in sog spatial expression, timing of activation and RNA Polymerase II loading rates. In addition, modeling of individual enhancer activities demonstrates that these enhancers integrate activation and repression signals differently. Whereas activation is due to the sum of the two enhancer activities, repression appears to depend on synergistic effects between enhancers. Finally, we examined the downstream signaling consequences resulting from the loss of either enhancer, and found changes in tissue patterning that can be explained by the differences in transcriptional kinetics measured.
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Proteínas de Drosophila , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica , Drosophila/metabolismo , GastrulaciónRESUMEN
A ubiquitous feature of the auditory organ in amniotes is the longitudinal mapping of neuronal characteristic frequencies (CFs), which increase exponentially with distance along the organ. The exponential tonotopic map reflects variation in hair cell properties according to cochlear location and is thought to stem from concentration gradients in diffusible morphogenic proteins during embryonic development. While in all amniotes the spatial gradient is initiated by sonic hedgehog (SHH), released from the notochord and floorplate, subsequent molecular pathways are not fully understood. In chickens, BMP7 is one such morphogen, secreted from the distal end of the cochlea. In mammals, the developmental mechanism differs from birds and may depend on cochlear location. A consequence of exponential maps is that each octave occupies an equal distance on the cochlea, a spacing preserved in the tonotopic maps in higher auditory brain regions. This may facilitate frequency analysis and recognition of acoustic sequences.
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Pollos , Proteínas Hedgehog , Animales , Pollos/metabolismo , Proteínas Hedgehog/metabolismo , Cóclea/metabolismo , Neuronas/metabolismo , Percepción , Mamíferos/metabolismoRESUMEN
Secreted signaling molecules act as morphogens to control patterning and growth in many developing tissues. Since locally produced morphogens spread to form a concentration gradient in the surrounding tissue, spreading is generally thought to be the key step in the non-autonomous actions. Here, we review recent advances in tool development to investigate morphogen function using the role of decapentaplegic (Dpp)/bone morphogenetic protein (BMP)-type ligand in the Drosophila wing disc as an example. By applying protein binder tools to distinguish between the roles of Dpp spreading and local Dpp signaling, we found that Dpp signaling in the source cells is important for wing patterning and growth but Dpp spreading from this source cells is not as strictly required as previously thought. Given recent studies showing unexpected requirements of long-range action of different morphogens, manipulating endogenous morphogen gradients by synthetic protein binder tools could shed more light on how morphogens act in developing tissues.