RESUMEN
An outstanding mystery in biology is why some species, such as the axolotl, can regenerate tissues whereas mammals cannot1. Here, we demonstrate that rapid activation of protein synthesis is a unique feature of the injury response critical for limb regeneration in the axolotl (Ambystoma mexicanum). By applying polysome sequencing, we identify hundreds of transcripts, including antioxidants and ribosome components that are selectively activated at the level of translation from pre-existing messenger RNAs in response to injury. By contrast, protein synthesis is not activated in response to non-regenerative digit amputation in the mouse. We identify the mTORC1 pathway as a key upstream signal that mediates tissue regeneration and translational control in the axolotl. We discover unique expansions in mTOR protein sequence among urodele amphibians. By engineering an axolotl mTOR (axmTOR) in human cells, we show that these changes create a hypersensitive kinase that allows axolotls to maintain this pathway in a highly labile state primed for rapid activation. This change renders axolotl mTOR more sensitive to nutrient sensing, and inhibition of amino acid transport is sufficient to inhibit tissue regeneration. Together, these findings highlight the unanticipated impact of the translatome on orchestrating the early steps of wound healing in a highly regenerative species and provide a missing link in our understanding of vertebrate regenerative potential.
Asunto(s)
Ambystoma mexicanum , Evolución Biológica , Biosíntesis de Proteínas , Regeneración , Serina-Treonina Quinasas TOR , Animales , Humanos , Ratones , Ambystoma mexicanum/fisiología , Secuencia de Aminoácidos , Extremidades/fisiología , Regeneración/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Cicatrización de Heridas , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Especificidad de la Especie , Antioxidantes/metabolismo , Nutrientes/metabolismo , Polirribosomas/genética , Polirribosomas/metabolismoRESUMEN
Ptch1 and Ptch2 are highly conserved vertebrate homologs of Drosophila ptc, the receptor of the Hedgehog (Hh) signaling pathway. The vertebrate Ptch1 gene encodes a potent tumor suppressor and is well established for its role in embryonic development. In contrast, Ptch2 is poorly characterized and dispensable for embryogenesis. In flies and mice, ptc/Ptch1 controls Hh signaling through the regulation of Smoothened (Smo). In addition, Hh pathway activation also up-regulates ptc/Ptch1 expression to restrict the diffusion of the ligand. Recent studies have implicated Ptch2 in this ligand dependent antagonism, however whether Ptch2 encodes a functional Shh receptor remains unclear. In this report, we demonstrate that Ptch2 is a functional Shh receptor, which regulates Smo localization and activity in vitro. We also show that Ptch1 and Ptch2 are co-expressed in the developing mouse limb bud and loss of Ptch2 exacerbates the outgrowth defect in the limb-specific Ptch1 knockout mutants, demonstrating that Ptch1 and Ptch2 co-operate in regulating cellular responses to Shh in vivo.
Asunto(s)
Extremidades/embriología , Morfogénesis/fisiología , Receptores de Superficie Celular/metabolismo , Animales , Western Blotting , Línea Celular , Proteínas Hedgehog/metabolismo , Hibridación in Situ , Ratones , Ratones Noqueados , Morfogénesis/genética , Receptores Patched , Receptor Patched-1 , Receptor Patched-2 , Receptores de Superficie Celular/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptor SmoothenedRESUMEN
BACKGROUND: The vertebrate digit pattern is defined by the morphogen Sonic hedgehog (Shh), which controls the activity of Gli transcription factors. Gli1, 2 and 3 are dynamically expressed during patterning. Downstream of Shh, their activity is regulated by Sufu and Kif7, core components of the Shh signaling cascade. The precise roles of these regulators during limb development have not been fully described. We analyze the role of Sufu and Kif7 in the limb and demonstrate that their loss has distinct and synergistic effects on Gli activity and digit pattern. RESULTS: Using a series of mouse mutants, we show that Sufu and Kif7 are expressed throughout limb development and their deletion has distinct effects on Gli levels and limb formation. Concomitant deletion of Sufu and Kif7 results in constitutive pathway activity and severe limb truncation. This is consistent with the recently published two-population model, which suggests that precocious activation of Shh signaling inhibits organizing center formation and limb outgrowth. CONCLUSIONS: Together, our findings demonstrate that perturbations of Sufu and Kif7 affect Gli activity and recapitulate the full spectrum of vertebrate limb defects, ranging from severe truncation to polydactyly.
Asunto(s)
Tipificación del Cuerpo/fisiología , Proteínas Hedgehog/metabolismo , Miembro Posterior/embriología , Cinesinas/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/fisiología , Animales , Proteínas Hedgehog/genética , Cinesinas/genética , Ratones , Ratones Noqueados , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Polidactilia/embriología , Polidactilia/genética , Proteínas Represoras/genética , Transactivadores/genética , Transactivadores/metabolismo , Proteína con Dedos de Zinc GLI1RESUMEN
Cells achieve highly efficient and accurate communication through cellular projections such as neurites and filopodia, yet there is a lack of genetically encoded tools that can selectively manipulate their composition and dynamics. Here, we present a versatile optogenetic toolbox of artificial multi-headed myosin motors that can move bidirectionally within long cellular extensions and allow for the selective transport of GFP-tagged cargo with light. Utilizing these engineered motors, we could transport bulky transmembrane receptors and organelles as well as actin remodellers to control the dynamics of both filopodia and neurites. Using an optimized in vivo imaging scheme, we further demonstrate that, upon limb amputation in axolotls, a complex array of filopodial extensions is formed. We selectively modulated these filopodial extensions and showed that they re-establish a Sonic Hedgehog signalling gradient during regeneration. Considering the ubiquitous existence of actin-based extensions, this toolbox shows the potential to manipulate cellular communication with unprecedented accuracy.
Asunto(s)
Comunicación Celular , Miosinas/metabolismo , Optogenética , Ingeniería de Proteínas , Citoesqueleto de Actina/metabolismo , Ambystoma mexicanum/fisiología , Animales , Transporte Biológico , Línea Celular , Supervivencia Celular/efectos de la radiación , Extremidades/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Hedgehog/metabolismo , Cinética , Luz , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Neuritas/metabolismo , Seudópodos/metabolismo , Regeneración/fisiología , Transducción de Señal , Vesículas Transportadoras/metabolismoRESUMEN
Although gene expression is tightly regulated during embryonic development, the impact of translational control has received less experimental attention. Here, we find that eukaryotic translation initiation factor-3 (eIF3) is required for Shh-mediated tissue patterning. Analysis of loss-of-function eIF3 subunit c (Eif3c) mice reveal a unique sensitivity to the Shh receptor patched 1 (Ptch1) dosage. Genome-wide in vivo enhanced cross-linking immunoprecipitation sequence (eCLIP-seq) shows unexpected specificity for eIF3 binding to a pyrimidine-rich motif present in subsets of 5'-UTRs and a corresponding change in the translation of these transcripts by ribosome profiling in Eif3c loss-of-function embryos. We further find a transcript specific effect in Eif3c loss-of-function embryos whereby translation of Ptch1 through this pyrimidine-rich motif is specifically sensitive to eIF3 amount. Altogether, this work uncovers hidden specificity of housekeeping translation initiation machinery for the translation of key developmental signaling transcripts.
Asunto(s)
Factor 3 de Iniciación Eucariótica/metabolismo , Biosíntesis de Proteínas/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Ribosomas/metabolismo , Animales , Línea Celular , Factor 3 de Iniciación Eucariótica/genética , Humanos , Ratones , ARN Mensajero/genética , Transducción de Señal/fisiologíaRESUMEN
In ribosomopathies, perturbed expression of ribosome components leads to tissue-specific phenotypes. What accounts for such tissue-selective manifestations as a result of mutations in the ribosome, a ubiquitous cellular machine, has remained a mystery. Combining mouse genetics and in vivo ribosome profiling, we observe limb-patterning phenotypes in ribosomal protein (RP) haploinsufficient embryos, and we uncover selective translational changes of transcripts that controlling limb development. Surprisingly, both loss of p53, which is activated by RP haploinsufficiency, and augmented protein synthesis rescue these phenotypes. These findings are explained by the finding that p53 functions as a master regulator of protein synthesis, at least in part, through transcriptional activation of 4E-BP1. 4E-BP1, a key translational regulator, in turn, facilitates selective changes in the translatome downstream of p53, and this thereby explains how RP haploinsufficiency may elicit specificity to gene expression. These results provide an integrative model to help understand how in vivo tissue-specific phenotypes emerge in ribosomopathies.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Extremidades/embriología , Haploinsuficiencia , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Ribosómicas/fisiología , Proteína p53 Supresora de Tumor/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Tipificación del Cuerpo , Proteínas de Ciclo Celular/genética , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Noqueados , Fenotipo , Ribosomas/metabolismoRESUMEN
The degree and dynamics of translational control during mammalian development remain poorly understood. Here we monitored translation of the mammalian genome as cells become specified and organize into tissues in vivo. This identified unexpected and pervasive translational regulation of most of the core signalling circuitry including Shh, Wnt, Hippo, PI3K and MAPK pathways. We further identify and functionally characterize a complex landscape of upstream open reading frames (uORFs) across 5'-untranslated regions (UTRs) of key signalling components. Focusing on the Shh pathway, we demonstrate the importance of uORFs within the major SHH receptor, Ptch1, in control of cell signalling and neuronal differentiation. Finally, we show that the expression of hundreds of mRNAs underlying critical tissue-specific developmental processes is largely regulated at the translation but not transcript levels. Altogether, this work reveals a new layer of translational control to major signalling components and gene regulatory networks that diversifies gene expression spatially across developing tissues.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Mamíferos/genética , Mamíferos/metabolismo , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/fisiología , Regiones no Traducidas 5'/genética , Regiones no Traducidas 5'/fisiología , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Femenino , Proteínas Hedgehog/metabolismo , Vía de Señalización Hippo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Células 3T3 NIH , Sistemas de Lectura Abierta/genética , Sistemas de Lectura Abierta/fisiología , Receptor Patched-1/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Alineación de Secuencia , Vía de Señalización WntRESUMEN
Hedgehog signaling is primarily transduced by two transcription factors: Gli2, which mainly acts as a full-length activator, and Gli3, which tends to be proteolytically processed from a full-length form (Gli3FL) to an N-terminal repressor (Gli3REP). Recent studies using a Sufu knockout mouse have indicated that Sufu is involved in regulating Gli2 and Gli3 activator and repressor activity at multiple steps of the signaling cascade; however, the mechanism of specific Gli2 and Gli3 regulation remains to be elucidated. In this study, we established an allelic series of ENU-induced mouse strains. Analysis of one of the missense alleles, SufuT396I, showed that Thr396 residue of Sufu played a key role in regulation of Gli3 activity. SufuT396I/T396I embryos exhibited severe polydactyly, which is indicative of compromised Gli3 activity. Concomitantly, significant quantitative reductions of unprocessed Gli3 (Gli3FL) and processed Gli3 (Gli3REP) were observed in vivo as well as in vitro. Genetic experiments showed that patterning defects in the limb buds of SufuT396I/T396I were rescued by a constitutive Gli3REP allele (Gli3∆699), strongly suggesting that SufuT396I reduced the truncated Gli3 repressor. In contrast, SufuT396I qualitatively exhibited no mutational effects on Gli2 regulation. Taken together, the results of this study show that the Thr396 residue of Sufu is specifically required for regulation of Gli3 but not Gli2. This implies a novel Sufu-mediated mechanism in which Gli2 activator and Gli3 repressor are differentially regulated.
Asunto(s)
Factores de Transcripción de Tipo Kruppel/química , Factores de Transcripción de Tipo Kruppel/metabolismo , Mutación Missense , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Animales , Tipificación del Cuerpo , Extremidades/crecimiento & desarrollo , Isoleucina/metabolismo , Ratones , Polidactilia/embriología , Polidactilia/genética , Estabilidad Proteica , Treonina/metabolismo , Proteína Gli2 con Dedos de Zinc , Proteína Gli3 con Dedos de ZincRESUMEN
The patterning and growth of the embryonic vertebrate limb is dependent on Sonic hedgehog (Shh), a morphogen that regulates the activity of Gli transcription factors. However, Shh expression is not observed during the first 12 hr of limb development. During this phase, the limb bud is prepatterned into anterior and posterior regions through the antagonistic actions of transcription factors Gli3 and Hand2. We demonstrate that precocious activation of Shh signaling during this early phase interferes with the Gli3-dependent specification of anterior progenitors, disturbing establishment of signaling centers and normal outgrowth of the limb. Our findings illustrate that limb development requires a sweet spot in the level and timing of pathway activation that allows for the Shh-dependent expansion of posterior progenitors without interfering with early prepatterning functions of Gli3/Gli3R or specification of anterior progenitors.