RESUMEN
Building a blastema from the stump is a key step of salamander limb regeneration. Stump-derived cells temporarily suspend their identity as they contribute to the blastema by a process generally referred to as dedifferentiation. Here, we provide evidence for a mechanism that involves an active inhibition of protein synthesis during blastema formation and growth. Relieving this inhibition results in a higher number of cycling cells and enhances the pace of limb regeneration. By small RNA profiling and fate mapping of skeletal muscle progeny as a cellular model for dedifferentiation, we find that the downregulation of miR-10b-5p is critical for rebooting the translation machinery. miR-10b-5p targets ribosomal mRNAs, and its artificial upregulation causes decreased blastema cell proliferation, reduction in transcripts that encode ribosomal subunits, diminished nascent protein synthesis, and retardation of limb regeneration. Taken together, our data identify a link between miRNA regulation, ribosome biogenesis, and protein synthesis during newt limb regeneration.
Asunto(s)
MicroARNs , ARN Pequeño no Traducido , Animales , Urodelos/genética , ARN Pequeño no Traducido/metabolismo , Músculo Esquelético/metabolismo , Ribosomas/genética , MicroARNs/genética , MicroARNs/metabolismo , Extremidades/fisiologíaRESUMEN
Salamanders exhibit an extraordinary ability among vertebrates to regenerate complex body parts. However, scarce genomic resources have limited our understanding of regeneration in adult salamanders. Here, we present the ~20 Gb genome and transcriptome of the Iberian ribbed newt Pleurodeles waltl, a tractable species suitable for laboratory research. We find that embryonic stem cell-specific miRNAs mir-93b and mir-427/430/302, as well as Harbinger DNA transposons carrying the Myb-like proto-oncogene have expanded dramatically in the Pleurodeles waltl genome and are co-expressed during limb regeneration. Moreover, we find that a family of salamander methyltransferases is expressed specifically in adult appendages. Using CRISPR/Cas9 technology to perturb transcription factors, we demonstrate that, unlike the axolotl, Pax3 is present and necessary for development and that contrary to mammals, muscle regeneration is normal without functional Pax7 gene. Our data provide a foundation for comparative genomic studies that generate models for the uneven distribution of regenerative capacities among vertebrates.
Asunto(s)
Extremidades/fisiología , Genoma/genética , MicroARNs/genética , Pleurodeles/genética , Regeneración/genética , Ambystoma mexicanum/genética , Animales , Sistemas CRISPR-Cas , Elementos Transponibles de ADN/genética , Células Madre Embrionarias/metabolismo , Edición Génica , Perfilación de la Expresión Génica , Genómica , Músculo Esquelético/fisiología , Factor de Transcripción PAX3/genética , Factor de Transcripción PAX7/genética , Proto-Oncogenes/genética , Regeneración/fisiologíaRESUMEN
Limb amputation in the newt induces myofibers to dedifferentiate and re-enter the cell cycle to generate proliferative myogenic precursors in the regeneration blastema. Here we show that bone morphogenetic proteins (BMPs) and mature BMPs that have been further cleaved by serum proteases induce cell cycle entry by dedifferentiating newt muscle cells. Protease-activated BMP4/7 heterodimers that are present in serum strongly induced myotube cell cycle re-entry with protease cleavage yielding a 30-fold potency increase of BMP4/7 compared with canonical BMP4/7. Inhibition of BMP signaling via muscle-specific dominant-negative receptor expression reduced cell cycle entry in vitro and in vivo. In vivo inhibition of serine protease activity depressed cell cycle re-entry, which in turn was rescued by cleaved-mimic BMP. This work identifies a mechanism of BMP activation that generates blastema cells from differentiated muscle.
Asunto(s)
Proteínas Morfogenéticas Óseas/farmacología , Ciclo Celular/efectos de los fármacos , Desdiferenciación Celular/efectos de los fármacos , Extremidades/fisiología , Células Musculares/citología , Péptido Hidrolasas/farmacología , Regeneración/efectos de los fármacos , Salamandridae/fisiología , Animales , Bovinos , Fibrinolisina/farmacología , Células HEK293 , Humanos , Células Musculares/efectos de los fármacos , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Multimerización de Proteína/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/farmacología , Fase S/efectos de los fármacos , Suero/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Smad/metabolismo , Trombina/farmacologíaRESUMEN
Caudal fin regeneration is characterized by a proliferation boost in the mesenchymal blastema that is controlled precisely in time and space. This allows a gradual and robust restoration of original fin size. However, how this is established and regulated is not well understood. Here, we report that Yap, the Hippo pathway effector, is a chief player in this process: functionally manipulating Yap during regeneration dramatically affects cell proliferation and expression of key signaling pathways, impacting regenerative growth. The intracellular location of Yap is tightly associated with different cell densities along the blastema proximal-distal axis, which correlate with alterations in cell morphology, cytoskeleton and cell-cell contacts in a gradient-like manner. Importantly, Yap inactivation occurs in high cell density areas, conditional to F-actin distribution and polymerization. We propose that Yap is essential for fin regeneration and that its function is dependent on mechanical tension, conferred by a balancing act of cell density and cytoskeleton activity.
