RESUMO
The presence of nerves is an important factor in successful organ regeneration in amphibians. The Mexican salamander, Ambystoma mexicanum, is able to regenerate limbs, tail, and gills when nerves are present. However, the nerve-dependency of tooth regeneration has not been evaluated. Here, we reevaluated tooth regeneration processes in axolotls using a three-dimensional reconstitution method called CoMBI and found that tooth regeneration is nerve-dependent although the dentary bone is independent of nerve presence. The induction and invagination of the dental lamina were delayed by denervation. Exogenous Fgf2, Fgf8, and Bmp7 expression could induce tooth placodes even in the denervated mandible. Our results suggest that the role of nerves is conserved and that Fgf+Bmp signals play key roles in axolotl organ-level regeneration. The presence of nerves is an important factor in successful organ regeneration in amphibians. The Mexican salamander, Ambystoma mexicanum, is able to regenerate limbs, tail, and gills when nerves are present. However, the nervedependency of tooth regeneration has not been evaluated. Here, we reevaluated tooth regeneration processes in axolotls using a three-dimensional reconstitution method called CoMBI and found that tooth regeneration is nerve-dependent although the dentary bone is independent of nerve presence. The induction and invagination of the dental lamina were delayed by denervation. Exogenous Fgf2, Fgf8, and Bmp7 expression could induce tooth placodes even in the denervated mandible. Our results suggest that the role of nerves is conserved and that Fgf+Bmp signals play key roles in axolotl organ-level regeneration.
Assuntos
Ambystoma mexicanum/fisiologia , Regeneração/fisiologia , Dente/fisiologia , Ambystoma mexicanum/genética , Animais , Animais Geneticamente Modificados , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 7/genética , Fator 2 de Crescimento de Fibroblastos/genética , Fator 8 de Crescimento de Fibroblasto/genética , Proteínas de Fluorescência Verde/genética , Proteínas Hedgehog/genética , Imageamento Tridimensional , Mandíbula/inervação , Mandíbula/cirurgia , Odontoblastos/citologia , Dente/anatomia & histologiaRESUMO
BACKGROUND: Urodele amphibians are capable of regenerating their organs after severe damage. During such regeneration, participating cells are given differentiation instructions by the surrounding cells. Limb regeneration has been investigated as a representative phenomenon of organ regeneration. Cells known as blastema cells are induced after limb amputation. In this process, dermal fibroblasts are dedifferentiated and become undifferentiated similar to limb bud cells. Just like limb bud cells, the induced blastema cells are positioned along the three limb developmental axes: the dorsoventral, the anteroposterior, and the proximodistal. The accurate developmental axes are essential for reforming the structures correctly. Despite the importance of the developmental axes, the relationship between the newly establishing developmental axes and existing limb axes was not well described with molecular markers. RESULTS: In this study, we grafted skin from GFP-transgenic axolotls and traced the cell lineage with position-specific gene expressions in order to investigate the correlation of the newly established axes and cellular origin. Shh- and Lmx1b-expressing cells emerged from the posterior skin and dorsal skin, respectively, even though the skin was transplanted to an inconsistent position. Shox2, a posterior marker gene, could be activated in cells derived from distal skin. CONCLUSIONS: Our results suggest that the location memories on anteroposterior and dorsoventral axes are relatively stable in a regenerating blastema though cellular differentiation is reprogrammed.
Assuntos
Ambystoma mexicanum/embriologia , Ambystoma mexicanum/fisiologia , Animais , Diferenciação Celular/fisiologia , Extremidades/embriologia , Extremidades/fisiologia , Imunofluorescência , Hibridização In Situ , Regeneração/fisiologiaRESUMO
Gill regeneration has not been well studied compared to regeneration of other appendages, such as limb and tail regeneration. Here, we focused on axolotl gill regeneration and found that Fgf- and Bmp-signaling are involved in their gill regeneration mechanism. Axolotls have three pairs of gill rami, and each gill ramus has multiple gill filaments. The gills consist of mesenchyme rich in extracellular matrix and epidermis. The gill nerves are supplied from the trigeminal ganglia located in the head. Denervation resulted in no gill regeneration responses. Nerves and gills express Bmp and Fgf genes, and treating animals with Fgf- and Bmp-signaling inhibitors results in phenotypes similar to those seen in denervated gills. Inducing an accessory appendage is a standard assay in amphibian regeneration research. In our study, an accessory gill could be induced by lateral wounding, suggesting that thin axon fibers and mesenchymal Fgfs and Bmps contributed to the induction of the accessory structure. Such accessory gill induction was inhibited by the denervation. Exogenous Fgf2+Fgf8+Bmp7, which have been determined to function as a regeneration inducer in urodele amphibians, could compensate for the effects denervation has on accessory blastema formation. Our findings suggest that regeneration of appendages in axolotls is regulated by common Fgf- and Bmp-signaling cascades.
Assuntos
Ambystoma mexicanum/metabolismo , Ambystoma mexicanum/fisiologia , Proteínas Morfogenéticas Ósseas/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Brânquias/fisiologia , Regeneração/fisiologia , Transdução de Sinais , Ambystoma mexicanum/genética , Animais , Proteínas Morfogenéticas Ósseas/genética , Denervação , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento , Brânquias/inervação , Organogênese/genética , Gânglio Trigeminal/metabolismoRESUMO
Urodele amphibians have a remarkable organ regeneration ability that is regulated by neural inputs. The identification of these neural inputs has been a challenge. Recently, Fibroblast growth factor (Fgf) and Bone morphogenic protein (Bmp) were shown to substitute for nerve functions in limb and tail regeneration in urodele amphibians. However, direct evidence of Fgf and Bmp being secreted from nerve endings and regulating regeneration has not yet been shown. Thus, it remained uncertain whether they were the nerve factors responsible for successful limb regeneration. To gather experimental evidence, the technical difficulties involved in the usage of axolotls had to be overcome. We achieved this by modifying the electroporation method. When Fgf8-AcGFP or Bmp7-AcGFP was electroporated into the axolotl dorsal root ganglia (DRG), GFP signals were detectable in the regenerating limb region. This suggested that Fgf8 and Bmp7 synthesized in neural cells in the DRG were delivered to the limbs through the long axons. Further knockdown experiments with double-stranded RNA interference resulted in impaired limb regeneration ability. These results strongly suggest that Fgf and Bmp are the major neural inputs that control the organ regeneration ability.
Assuntos
Ambystoma mexicanum/fisiologia , Proteína Morfogenética Óssea 7/genética , Extremidades/fisiologia , Fatores de Crescimento de Fibroblastos/genética , Regeneração/fisiologia , Animais , Proteína Morfogenética Óssea 7/metabolismo , Células Cultivadas , Fatores de Crescimento de Fibroblastos/metabolismo , Gânglios Espinais/citologia , Gânglios Espinais/crescimento & desenvolvimento , Proteínas de Fluorescência Verde/genética , Hibridização in Situ Fluorescente , Tecido Nervoso/metabolismo , Neurônios/metabolismo , Reação em Cadeia da Polimerase , Interferência de RNA , RNA Interferente Pequeno/genética , CaudaRESUMO
Amphibians can regenerate missing body parts, including limbs. The regulation of collagen has been considered to be important in limb regeneration. Collagen deposition is suppressed during limb regeneration, so we investigated collagen deposition and apical epithelial cap (AEC) formation during axolotl limb regeneration. The accessory limb model (ALM) has been developed as an alternative model for studying limb regeneration. Using this model, we investigated the relationship between nerves, epidermis, and collagen deposition. We found that Sp-9, an AEC marker gene, was upregulated by direct interaction between nerves and epidermis. However, collagen deposition hindered this interaction, and resulted in the failure of limb regeneration. During wound healing, an increase in deposition of collagen caused a decrease in the blastema induction rate in ALM. Wound healing and limb regeneration are alternate processes.
Assuntos
Ambystoma mexicanum/fisiologia , Colágeno/fisiologia , Regeneração/fisiologia , Animais , Extremidades/inervação , Extremidades/fisiologia , Botões de Extremidades/fisiologia , Pele/inervação , Fenômenos Fisiológicos da PeleRESUMO
Urodele amphibians can regenerate amputated limbs. It has been considered that differentiated dermal tissues generate multipotent and undifferentiated cells called blastema cells during limb regeneration. In early phases of limb regeneration, blastema cells are induced by nerves and the apical epithelial cap (AEC). We had previously investigated the role of neurotrophic factors in blastema or blastema-like formation consisting of Prrx-1 positive cells. A new system suitable for investigating early phases of limb regeneration, called the accessory limb model (ALM), was recently developed. In this study, we performed a comparative transcriptome analysis between a blastema and wound using ALM. Matrix metalloproteinase (MMP) and fibroblast growth factor (FGF) signaling components were observed to be predominantly expressed in ALM blastema cells. Furthermore, we found that MMP activity induced a blastema marker gene, Prrx-1, in vitro, and FGF signaling pathways worked in coordination to maintain Prrx-1 expression and ALM blastema formation. Furthermore, we demonstrated that these two activities were sufficient to induce an ALM blastema in the absence of a nerve in vivo.