Limb Regeneration Process Research Articles

The Impact of Senescent Cells on Limb Regeneration.

Cellular senescence is a state in which cells enter cell cycle arrest. However, senescent cells have the ability to secrete signaling molecules such as chemokines, cytokines, and growth factors. This secretory activity is an important feature of senescent cells, since the secreted factors impact the surrounding cellular microenvironment. Indeed, senescent cells and their secretome play a crucial role during limb development. However, whether the process of limb regeneration also relies on senescent cells remains unclear. Creation of a novel targeted depletion strategy that can eliminate senescent cells in the regenerating limb has now demonstrated an important role for senescent cells in limb regeneration. This role is linked to senescent cell-derived Wnt signaling. These findings reveal a previously unknown role for senescent cells during limb regeneration through Wnt signaling.

Morphological and Molecular Changes during Limb Regeneration of the Exopalaemon carinicauda.

With the increase in breeding density of Exopalaemon carinicauda, appendage breakage may occur, which seriously affects survival and economic benefits. To study the limb regeneration process of E. carinicauda, we induced autotomy of the pereopods. After a period of time, wound swelling disappeared, the pigment gradually accumulated, and a tawny film subsequently formed in the wound. The healing period of the wound occurred 24 h after autotomy, and the blastema formation stage occurred 48 h after autotomy. After 4 days of cutting, the limb buds began to differentiate, grow, and expand rapidly, and this process lasted approximately 15 days. Microscopic observations revealed significant changes in the type and number of associated cells including outer epithelial cells, granulocytes, embryonic cells, columnar epidermal cells, elongated cells, and blastoma cells, during the process from limb fracture to regeneration. A comparative transcriptome analysis identified 1415 genes differentially expressed between the J0h (0 h post autotomy) and J18h (18 h post autotomy), and 3952 and 4366 differentially expressed genes for J0 and J14d (14 days post autotomy) and J18h and J14d, respectively. Some of these genes may be related to muscle growth or molting, as indicated by the presence of troponin C, chitinase, actin, innexin, and cathepsin L. As a functional gene involved in epidermal formation, the mRNA expression level of the innexin inx2 in the pereopod of E. carinicauda changed significantly in the experimental groups (p < 0.05). The results of this study contribute to existing knowledge of regeneration mechanisms in crustaceans.

Adaptation of Glucose Metabolism to Limb Autotomy and Regeneration in the Chinese Mitten Crab.

Limb autotomy and regeneration represent distinctive responses of crustaceans to environmental stress. Glucose metabolism plays a pivotal role in energy generation for tissue development and regeneration across various species. However, the relationship between glucose metabolism and tissue regeneration in crustaceans remains elusive. Therefore, this study is aimed at analyzing the alterations of glucose metabolic profile during limb autotomy and regeneration in Eriocheir sinensis, while also evaluating the effects of carbohydrate supplementation on limb regeneration. The results demonstrated that limb autotomy triggered a metabolic profile adaption at the early stage of regeneration. Hemolymph glucose levels were elevated, and multiple glucose catabolic pathways were enhanced in the hepatopancreas. Additionally, glucose and ATP levels in the regenerative limb were upregulated, along with increased expression of glucose transporters. Furthermore, the gene expression and activity of enzymes involved in gluconeogenesis were repressed in the hepatopancreas. These findings indicate that limb regeneration triggers metabolic profile adaptations to meet the elevated energy requirements. Moreover, the study observed that supplementation with corn starch enhanced limb regeneration capacity by promoting wound healing and blastema growth. Interestingly, dietary carbohydrate addition influenced limb regeneration by stimulating gluconeogenesis rather than glycolysis in the regenerative limb. Thus, these results underscore the adaptation of glucose metabolism during limb autotomy and regeneration, highlighting its essential role in the limb regeneration process of E. sinensis.

Transcriptome analysis of Litopenaeus vannamei during the early stage of limb regeneration process

Regeneration is a process in which organisms regrow new tissues or organs at the injury site, which has attracted the attention of many scientists and nonscientists. However, the underlying molecular mechanisms of regeneration after autotomy are largely unknown. In this study, we conducted RNA-seq sequencing on regenerated limb bud tissues of Litopenaeus vannamei at 0 hours post autotomy (0 hpa), 12 hours post autotomy (12 hpa), and 24 hours post autotomy (24 hpa). A total of 2,192 differentially expressed genes related to energy metabolism, transcription and translation, and epidermis development were identified between 0 hpa and 12 hpa, such as triosephosphate isomerase A, triosephosphate isomerase B, and zinc finger protein 367 that is upregulated in 12 hpa. Between 12 hpa and 24 hpa, 1,447 differentially expressed genes were identified that were related to cuticle development and energy metabolism, such as cuticle protein 6, which is upregulated in 24 hpa, and triosephosphate isomerase is downregulated in 24 hpa. The results indicated that energy metabolism, transcription and translation, epidermal formation, and chitin metabolism processes are involved during the early stage of limb regeneration. This study provides basic knowledge for investigating the molecular mechanisms associated with limb regeneration in crustaceans at the early regeneration stage.

En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration.

The mouse digit tip regenerates following amputation. How the regenerate is patterned is unknown, but a long-standing hypothesis proposes developmental patterning mechanisms are re-used during regeneration. The digit tip bone exhibits dorsal-ventral (DV) polarity, so we focus on En1 and Lmx1b, two factors necessary for DV patterning during limb development. We investigate whether they are re-expressed during regeneration in a developmental-like pattern and whether they direct DV morphology of the regenerate. We find that both En1 and Lmx1b are expressed in the regenerating digit tip epithelium and mesenchyme, respectively, but without DV polarity. Conditional genetics and quantitative analysis of digit tip bone morphology determine that genetic deletion of En1 or Lmx1b in adult digit tip regeneration modestly reduces bone regeneration but does not affect DV patterning. Collectively, our data suggest that, while En1 and Lmx1b are re-expressed during mouse digit tip regeneration, they do not define the DV axis during regeneration.

"Omics" data unveil early molecular response underlying limb regeneration in the Chinese mitten crab, Eriocheir sinensis.

Limb regeneration is a fascinating and medically interesting trait that has been well preserved in arthropod lineages, particularly in crustaceans. However, the molecular mechanisms underlying arthropod limb regeneration remain largely elusive. The Chinese mitten crab Eriocheir sinensis shows strong regenerative capacity, a trait that has likely allowed it to become a worldwide invasive species. Here, we report a chromosome-level genome of E. sinensis as well as large-scale transcriptome data during the limb regeneration process. Our results reveal that arthropod-specific genes involved in signal transduction, immune response, histone methylation, and cuticle development all play fundamental roles during the regeneration process. Particularly, Innexin2-mediated signal transduction likely facilitates the early stage of the regeneration process, while an effective crustacean-specific prophenoloxidase system (ProPo-AS) plays crucial roles in the initial immune response. Collectively, our findings uncover novel genetic pathways pertaining to arthropod limb regeneration and provide valuable resources for studies on regeneration from a comparative perspective.

The effects of feeding ration and cheliped autotomy on the growth and expression of ecdysteroid receptor in early juvenile mud crabs, Scylla paramamosain

This study investigated the combined effects of feeding ration and cheliped autotomy on the intermolt duration, molting success, molt increments in size and weight, and ecdysteroid receptor gene (SpEcR) expression of early juvenile mud crab Scylla paramamosain. Newly molted second stage juvenile crabs (C2) were subjected to four feeding conditions; optimal, suboptimal (1/2 optimal), low (1/4 optimal) ration and starvation, and autotomy (intact vs. cheliped autotomy) in a 4 × 2 factorial design until all crabs successfully molted or died. A significant interaction of feeding ration and cheliped autotomy on intermolt duration was identified. With reduced feeding ration, both intact and cheliped autotomized crabs showed increased time and desynchrony of molting, but decreased carapace size and body weight. Importantly, all crabs with different feeding rations even the low ration had high rates of molting success (> 95%), while the crabs subjected to starvation died without molting. When fed optimal ration, the mean intermolt duration of the cheliped autotomized crabs was significantly prolonged, while no such effect was found between autotomized and intact crabs subjected to suboptimal or low feeding ration. The qRT-PCR revealed that the expression of SpEcR showed a general trend of inhibited by reduced feeding ration, which was consistent with observed significantly increased intermolt duration. Interestingly, the transcript level of SpEcR was only significantly affected by cheliped autotomy under the optimal and suboptimal feeding rations but not for the low feeding ration. Together, the results of this study suggest that the S. paramamosain early juveniles have a strong tolerance for fluctuations in food availability. In addition, the availability of food and limb autotomy could significantly affect growth, molting duration and synchrony of the crabs, which appeared to reflect in SpEcR expression level that involved in the regulation of molting and limb regeneration process of the juvenile crabs.

Lmx1b activation in axolotl limb regeneration.

Axolotls can regenerate their limbs. In their limb regeneration process, developmental genes are re-expressed and reorganize the developmental axes, in which the position-specific genes are properly re-expressed. However, how such position specificity is reorganized in the regeneration processes has not been clarified. To address this issue, we focused on the reactivation process of Lmx1b, which determines the limb dorsal identity in many animals. Here, we show that Lmx1b expression is maintained in the dorsal skin before amputation and is activated after amputation. Furthermore, we demonstrate that only cells located in the dorsal side prior to limb amputation could reactivate Lmx1b after limb amputation. We also found that Lmx1b activation was achieved by nerve presence. The nerve factors, BMP2+FGF2+FGF8 (B2FF), consistently reactivate Lmx1b when applied to the dorsal skin. These results imply that the retained Lmx1b expression in the intact skin plays a role in positional memory, which instruct cells about the spatial positioning before amputation. This memory is reactivated by nerves or nerve factors that can trigger the entire limb regeneration process. Our findings highlight the role of nerves in amphibian limb regeneration, including both the initiation of limb regeneration and the reactivation of position-specific gene expression.

DNA repair during regeneration in Ambystoma mexicanum.

The remarkable regenerative capabilities of the salamander Ambystoma mexicanum have turned it into one of the principal models to study limb regeneration. During this process, a mass of low differentiated and highly proliferative cells, called blastema, propagates to reestablish the lost tissue in an accelerated way. Such a process implies the replication of a huge genome, 10 times larger than humans, with about 65.6% of repetitive sequences. These features make the axolotl genome inherently difficult to replicate and prone to bear mutations. In this context, the role of DNA repair mechanisms acquires great relevance to maintain genomic stability, especially if we consider the necessity of ensuring the correct replication and integrity of such a large genome in the blastema cells, which are key for tissue regeneration. On the contrary, DNA damage accumulation in these cells may result in senescence, apoptosis and premature differentiation, all of them are mechanisms employed to avoid DNA damage perpetuation but with the potential to affect the limb regeneration process. Here we review and discuss the current knowledge on the implications of DNA damage responses during salamander regeneration.

Zebrafish (Danio rerio) as a Model for Understanding the Process of Caudal Fin Regeneration.

After its introduction for scientific investigation in the 1950s, the cypriniform zebrafish, Danio rerio, has become a valuable model for the study of regenerative processes and mechanisms. Zebrafish exhibit epimorphic regeneration, in which a nondifferentiated cell mass formed after amputation is able to fully regenerate lost tissue such as limbs, heart muscle, brain, retina, and spinal cord. The process of limb regeneration in zebrafish comprises several stages characterized by the activation of specific signaling pathways and gene expression. We review current research on key factors in limb regeneration using zebrafish as a model.

Single-cell RNA-seq reveals novel mitochondria-related musculoskeletal cell populations during adult axolotl limb regeneration process.

While the capacity to regenerate tissues or limbs is limited in mammals, including humans, axolotls are able to regrow entire limbs and major organs after incurring a wound. The wound blastema has been extensively studied in limb regeneration. However, due to the inadequate characterization of ECM and cell subpopulations involved in the regeneration process, the discovery of the key drivers for human limb regeneration remains unknown. In this study, we applied large-scale single-cell RNA sequencing to classify cells throughout the adult axolotl limb regeneration process, uncovering a novel regeneration-specific mitochondria-related cluster supporting regeneration through energy providing and the ECM secretion (COL2+) cluster contributing to regeneration through cell-cell interactions signals. We also discovered the dedifferentiation and re-differentiation of the COL1+/COL2+ cellular subpopulation and exposed a COL2-mitochondria subcluster supporting the musculoskeletal system regeneration. On the basis of these findings, we reconstructed the dynamic single-cell transcriptome of adult axolotl limb regenerative process, and identified the novel regenerative mitochondria-related musculoskeletal populations, which yielded deeper insights into the crucial interactions between cell clusters within the regenerative microenvironment.

New insight into functional limb regeneration: A to Z approaches.

Limb/digit amputation is a common event in humans caused by trauma, medical illness, or surgery. Although the loss of a digit is not lethal, it affects quality of life and imposes high costs on amputees. In recent years, the increasing interest in limb regeneration has led to enhanced scientific knowledge. However, the limited ability to develop functional limb regeneration in the clinical setting suggests that a challenging issue remains in limb regeneration. Recently, the emergence of regenerative engineering is a promising field to address this challenge and close the gap between science and clinical applications. Cell signalling and molecular mechanisms involved in the limb regeneration process have been extensively studied; however, there is still insufficient data on cell therapy and tissue engineering for limb regeneration. In this review, we intend to focus on therapeutic approaches for limb regeneration that are closely related to gene, immune, and stem cell therapies, as well as tissue engineering approaches that take into consideration the peculiar developmental properties of the limbs. In addition, we attempt to identify the challenges of these strategies for limb regeneration studies in terms of clinical settings and as a road map to accomplish the goal of functional human limb regeneration.

Profiling of glycan alterations in regrowing limb tissues of Cynops orientalis.

Glycans are known to play important roles in molecular recognition, cell-cell adhesion, molecular trafficking, receptor activation, and signal transduction during development and regeneration. Despite numerous investigations of regenerating salamander limbs, global analysis of the precise variation of glycans during the limb regeneration process has received little attention. Here, we have used lectin microarrays and lectin histochemistry to analyze the alterations and distribution of glycans during the early stages leading to blastema formation during Cynops orientalis limb regeneration in response to limb amputation. Compared with the control group, analysis at several time points (3, 7, and 14 days postamputation) using microarrays containing 37 lectins showed that limb tissues expressed significantly different complements of glycans recognized by 9 different lectins. Postamputation limb tissues showed higher expression of two glycan structures recognized by the lectins STL and LTL and lower expression of seven glycan structures recognized by PHA-E, MAL-I, SNA, UEA-I, PHA-E + L, VVA, and GNA. We also observed significant changes in glycans specifically at 7 days postamputation, including higher binding capacity by WFA, GSL-I, and NPA and lower binding capacity by PNA, HHL, ConA, LCA, GSL-II, and PWM. Next, we validated our lectin microarray data using lectin histochemistry in limb tissues. Glycans recognized by STL and GNA showed similar changes in signal intensity to those found in the lectin microarrays, with STL staining in the cytoplasm and GNA in the cytoplasm and nucleus. Our findings are the first report of significant postamputation changes in glycans in limb tissues and suggest that those glycans perform potentially important functions during the early stages of C. orientalis limb regeneration.

Limb regeneration in a direct-developing terrestrial salamander, Bolitoglossa ramosi (Caudata: Plethodontidae): Limb regeneration in plethodontid salamanders.

Appendage regeneration is one of the most compelling phenomena in regenerative biology and is extensively studied in axolotls and newts. However, the regenerative capacity in other families of salamanders remains poorly described. Here we characterize the limb regeneration process in Bolitoglossa ramosi, a direct‐developing terrestrial salamander of the plethodontid family. We (1) describe the major morphological features at different stages of limb regeneration, (2) show that appendage regeneration in a terrestrial salamander varies from other amphibians and (3) show that limb regeneration in this species is considerably slower than in axolotls and newts (95 days post‐amputation for complete regeneration) despite having a significantly smaller genome size than axolotls or newts.

The Role of the Prod1 Membrane Anchor in Newt Limb Regeneration.

Prod1 is a protein that regulates limb regeneration in salamanders by determining the direction of limb growth. Prod1 is attached to the membrane by a glycosylphosphatidylinositol (GPI) anchor, but the role of membrane anchoring in the limb regeneration process is poorly understood. In this study, we investigated the functional role of the anchoring of Prod1 to the membrane by using its synthetic mimics in combination with solid‐state NMR spectroscopy and fluorescent microscopy techniques. Anchoring did not affect the three‐dimensional structure of Prod1 but did induce aggregation by aligning the molecules and drastically reducing the molecular motion on the two‐dimensional membrane surface. Interestingly, aggregated Prod1 interacted with Prod1 molecules tethered on the surface of opposing membranes, inducing membrane adhesion. Our results strongly suggest that anchoring of the salamander‐specific protein Prod1 assists cell adhesion in the limb regeneration process.

The Role of the Prod1 Membrane Anchor in Newt Limb Regeneration

AbstractProd1 is a protein that regulates limb regeneration in salamanders by determining the direction of limb growth. Prod1 is attached to the membrane by a glycosylphosphatidylinositol (GPI) anchor, but the role of membrane anchoring in the limb regeneration process is poorly understood. In this study, we investigated the functional role of the anchoring of Prod1 to the membrane by using its synthetic mimics in combination with solid‐state NMR spectroscopy and fluorescent microscopy techniques. Anchoring did not affect the three‐dimensional structure of Prod1 but did induce aggregation by aligning the molecules and drastically reducing the molecular motion on the two‐dimensional membrane surface. Interestingly, aggregated Prod1 interacted with Prod1 molecules tethered on the surface of opposing membranes, inducing membrane adhesion. Our results strongly suggest that anchoring of the salamander‐specific protein Prod1 assists cell adhesion in the limb regeneration process.

Limbform: a functional ontology-based database of limb regeneration experiments.

The ability of certain organisms to completely regenerate lost limbs is a fascinating process, far from solved. Despite the extraordinary published efforts during the past centuries of scientists performing amputations, transplantations and molecular experiments, no mechanistic model exists yet that can completely explain patterning during the limb regeneration process. The lack of a centralized repository to enable the efficient mining of this huge dataset is hindering the discovery of comprehensive models of limb regeneration. Here, we introduce Limbform (Limb formalization), a centralized database of published limb regeneration experiments. In contrast to natural language or text-based ontologies, Limbform is based on a functional ontology using mathematical graphs to represent unambiguously limb phenotypes and manipulation procedures. The centralized database currently contains >800 published limb regeneration experiments comprising many model organisms, including salamanders, frogs, insects, crustaceans and arachnids. The database represents an extraordinary resource for mining the existing knowledge of functional data in this field; furthermore, its mathematical nature based on a functional ontology will pave the way for artificial intelligence tools applied to the discovery of the sought-after comprehensive limb regeneration models.

Understanding positional cues in salamander limb regeneration: implications for optimizing cell-based regenerative therapies.

Regenerative medicine has reached the point where we are performing clinical trials with stem-cell-derived cell populations in an effort to treat numerous human pathologies. However, many of these efforts have been challenged by the inability of the engrafted populations to properly integrate into the host environment to make a functional biological unit. It is apparent that we must understand the basic biology of tissue integration in order to apply these principles to the development of regenerative therapies in humans. Studying tissue integration in model organisms, where the process of integration between the newly regenerated tissues and the ‘old’ existing structures can be observed and manipulated, can provide valuable insights. Embryonic and adult cells have a memory of their original position, and this positional information can modify surrounding tissues and drive the formation of new structures. In this Review, we discuss the positional interactions that control the ability of grafted cells to integrate into existing tissues during the process of salamander limb regeneration, and discuss how these insights could explain the integration defects observed in current cell-based regenerative therapies. Additionally, we describe potential molecular tools that can be used to manipulate the positional information in grafted cell populations, and to promote the communication of positional cues in the host environment to facilitate the integration of engrafted cells. Lastly, we explain how studying positional information in current cell-based therapies and in regenerating limbs could provide key insights to improve the integration of cell-based regenerative therapies in the future.

Microarray Analysis of microRNA Expression during Axolotl Limb Regeneration

Among vertebrates, salamanders stand out for their remarkable capacity to quickly regrow a myriad of tissues and organs after injury or amputation. The limb regeneration process in axolotls (Ambystoma mexicanum) has been well studied for decades at the cell-tissue level. While several developmental genes are known to be reactivated during this epimorphic process, less is known about the role of microRNAs in urodele amphibian limb regeneration. Given the compelling evidence that many microRNAs tightly regulate cell fate and morphogenetic processes through development and adulthood by modulating the expression (or re-expression) of developmental genes, we investigated the possibility that microRNA levels change during limb regeneration. Using two different microarray platforms to compare the axolotl microRNA expression between mid-bud limb regenerating blastemas and non-regenerating stump tissues, we found that miR-21 was overexpressed in mid-bud blastemas compared to stump tissue. Mature A. mexicanum (“Amex”) miR-21 was detected in axolotl RNA by Northern blot and differential expression of Amex-miR-21 in blastema versus stump was confirmed by quantitative RT-PCR. We identified the Amex Jagged1 as a putative target gene for miR-21 during salamander limb regeneration. We cloned the full length 3′UTR of Amex-Jag1, and our in vitro assays demonstrated that its single miR-21 target recognition site is functional and essential for the response of the Jagged1 gene to miR-21 levels. Our findings pave the road for advanced in vivo functional assays aimed to clarify how microRNAs such as miR-21, often linked to pathogenic cell growth, might be modulating the redeployment of developmental genes such as Jagged1 during regenerative processes.

Lessons for Adult Fingertip Regeneration: Glimpses From Basic Research

Understanding the mechanisms involved in limb and finger regeneration holds promise for improving current treatment therapies. Recent animal studies have improved our understanding of the limb regeneration process markedly. Improved sophistication in experimentation has allowed results that partly reveal the cells of origin in fingertip regeneration in mouse models, which implicates a tissue-resident progenitor cell population. The impressive regeneration of amputated salamander limbs has been shown to work through an evolutionarily divergent mechanism and may not be open to direct translational approaches in mammals. In addition, researchers are beginning to understand the complexity of the interrelated mechanisms of axis determinants in chick embryo limb development. In this article, we review lessons to be learned from these divergent experiments, to understand fingertip regeneration in humans.