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Nerve presence in the axolotl mandible and a regenerating tooth. βIII tubulin-GFP transgenic axolotl tissues show the presence of axons. (A-C) GFP signals in normal (control) axolotl mandible. (A) Lateral view. (B) Ventral view. (C) Dorsal view of the mandible. Subsequently, the lower jaw was dissected out and pictures were taken from the dorsal side. (D-F) Nerve presence in the regenerating tooth. GFP positive nerve fibers were visualized by anti-GFP antibody on the sections. D'-F' are higher magnification views of the boxed regions in D-F. (G-I) Nerve presence in the regenerating tooth in the denervated mandible. G'-I' are higher magnification views of the boxed regions in G-I. Scale bars in A, B, and C are 5, 5, 1. Scale bars in D-I and D'-I' are 0.3 and 0.05 mm, respectively. The dotted lines indicate the border of the oral epithelium. Data are representative of 4 independent experiments.
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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 usi...
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... dependency of axolotl tooth regeneration. To examine the structures of nerves in detail, we used βIII-tubulin GFP transgenic axolotls, which are convenient for monitoring axon presence in tissues 24 . Axons projecting to the mandible could be observed in the smaller specimens (Fig. 5). Axons projecting from the trigeminal ganglia toward the mandible were also visible ( Fig. 5A-C). The nerves running into the ventral root exhibit a complex nerve projection pattern. In the proximal region of the dentary, the nerves branch apart into two major routes. In a βIII-tubulin GFP transgenic axolotl, GFP-positive fibers could ...
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... regeneration. To examine the structures of nerves in detail, we used βIII-tubulin GFP transgenic axolotls, which are convenient for monitoring axon presence in tissues 24 . Axons projecting to the mandible could be observed in the smaller specimens (Fig. 5). Axons projecting from the trigeminal ganglia toward the mandible were also visible ( Fig. 5A-C). The nerves running into the ventral root exhibit a complex nerve projection pattern. In the proximal region of the dentary, the nerves branch apart into two major routes. In a βIII-tubulin GFP transgenic axolotl, GFP-positive fibers could be confirmed in the www.nature.com/scientificreports www.nature.com/scientificreports/ ...
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... into the ventral root exhibit a complex nerve projection pattern. In the proximal region of the dentary, the nerves branch apart into two major routes. In a βIII-tubulin GFP transgenic axolotl, GFP-positive fibers could be confirmed in the www.nature.com/scientificreports www.nature.com/scientificreports/ regenerating axolotl mandible on day 15 (Fig. 5D,D'). A few GFP-positive cells were identifiable in both the mesenchyme and the oral epithelium. The GFP signal was increased on day 30 (Fig. 5E,E'), and GFP signals were still found in the regenerated tooth on day 45 (Fig. 4F,F'). Notably, GFP positive axons could be seen in the epithelium adjacent to the forming tooth bud (Fig. 5E,E' ...
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... routes. In a βIII-tubulin GFP transgenic axolotl, GFP-positive fibers could be confirmed in the www.nature.com/scientificreports www.nature.com/scientificreports/ regenerating axolotl mandible on day 15 (Fig. 5D,D'). A few GFP-positive cells were identifiable in both the mesenchyme and the oral epithelium. The GFP signal was increased on day 30 (Fig. 5E,E'), and GFP signals were still found in the regenerated tooth on day 45 (Fig. 4F,F'). Notably, GFP positive axons could be seen in the epithelium adjacent to the forming tooth bud (Fig. 5E,E' ,F,F'). This implies a positive relationship between tooth bud initiation and nerves. We next investigated the roles of nerves in tooth ...
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... on day 15 (Fig. 5D,D'). A few GFP-positive cells were identifiable in both the mesenchyme and the oral epithelium. The GFP signal was increased on day 30 (Fig. 5E,E'), and GFP signals were still found in the regenerated tooth on day 45 (Fig. 4F,F'). Notably, GFP positive axons could be seen in the epithelium adjacent to the forming tooth bud (Fig. 5E,E' ,F,F'). This implies a positive relationship between tooth bud initiation and nerves. We next investigated the roles of nerves in tooth regeneration through denervation experiments. Our denervation procedure targeted the two major nerve routes in the mandibular region ( Fig. 5A-C). In the first denervation, the proximal region of ...
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... could be seen in the epithelium adjacent to the forming tooth bud (Fig. 5E,E' ,F,F'). This implies a positive relationship between tooth bud initiation and nerves. We next investigated the roles of nerves in tooth regeneration through denervation experiments. Our denervation procedure targeted the two major nerve routes in the mandibular region ( Fig. 5A-C). In the first denervation, the proximal region of each branch was dissected (Fig. 5A,B). Dentectomy was performed on the same day as the first denervation. Knowing that newly regenerating axons emerge from the dissected ends of nerves and that these newly forming axons are invisible because of their thinness, we also performed a ...
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... implies a positive relationship between tooth bud initiation and nerves. We next investigated the roles of nerves in tooth regeneration through denervation experiments. Our denervation procedure targeted the two major nerve routes in the mandibular region ( Fig. 5A-C). In the first denervation, the proximal region of each branch was dissected (Fig. 5A,B). Dentectomy was performed on the same day as the first denervation. Knowing that newly regenerating axons emerge from the dissected ends of nerves and that these newly forming axons are invisible because of their thinness, we also performed a second denervation (on day 10) on the more basal region (Fig. 5A). In the denervated mandible, ...
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... region of each branch was dissected (Fig. 5A,B). Dentectomy was performed on the same day as the first denervation. Knowing that newly regenerating axons emerge from the dissected ends of nerves and that these newly forming axons are invisible because of their thinness, we also performed a second denervation (on day 10) on the more basal region (Fig. 5A). In the denervated mandible, GFP signals were almost absent initially (Fig. 5G,G'). By day 30, however, a few GFP-positive fibers could be seen (Fig. 5H,H'). On day 45, the axon presence remained much lower in denervated mandibles than in control mandibles (Fig. 5F,F' ,I,I'). These results indicate that our denervation procedure ...
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... same day as the first denervation. Knowing that newly regenerating axons emerge from the dissected ends of nerves and that these newly forming axons are invisible because of their thinness, we also performed a second denervation (on day 10) on the more basal region (Fig. 5A). In the denervated mandible, GFP signals were almost absent initially (Fig. 5G,G'). By day 30, however, a few GFP-positive fibers could be seen (Fig. 5H,H'). On day 45, the axon presence remained much lower in denervated mandibles than in control mandibles (Fig. 5F,F' ,I,I'). These results indicate that our denervation procedure results in an aneurogenic state in the early phase but that innervation is somehow ...
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... emerge from the dissected ends of nerves and that these newly forming axons are invisible because of their thinness, we also performed a second denervation (on day 10) on the more basal region (Fig. 5A). In the denervated mandible, GFP signals were almost absent initially (Fig. 5G,G'). By day 30, however, a few GFP-positive fibers could be seen (Fig. 5H,H'). On day 45, the axon presence remained much lower in denervated mandibles than in control mandibles (Fig. 5F,F' ,I,I'). These results indicate that our denervation procedure results in an aneurogenic state in the early phase but that innervation is somehow recovered in the later ...
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... we also performed a second denervation (on day 10) on the more basal region (Fig. 5A). In the denervated mandible, GFP signals were almost absent initially (Fig. 5G,G'). By day 30, however, a few GFP-positive fibers could be seen (Fig. 5H,H'). On day 45, the axon presence remained much lower in denervated mandibles than in control mandibles (Fig. 5F,F' ,I,I'). These results indicate that our denervation procedure results in an aneurogenic state in the early phase but that innervation is somehow recovered in the later ...
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... in teeth has been conserved widely among species. Our study revealed the presence of axons in teeth in the axolotl mandible. Axons were observed in the pre-invagination oral epithelium (Fig. 5E,F), the developing tooth buds (Fig. 2I,J), and the mature tooth (Fig. 2K). The axons originate from the trigeminal ganglia (Fig. 5A-C), then from many branches in the proximal region of the dentary (Fig. 5A-C). Our denervation procedure targeted major routes but was not expected to remove all of them. Insufficient removal of axons or axon ...
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... in teeth has been conserved widely among species. Our study revealed the presence of axons in teeth in the axolotl mandible. Axons were observed in the pre-invagination oral epithelium (Fig. 5E,F), the developing tooth buds (Fig. 2I,J), and the mature tooth (Fig. 2K). The axons originate from the trigeminal ganglia (Fig. 5A-C), then from many branches in the proximal region of the dentary (Fig. 5A-C). Our denervation procedure targeted major routes but was not expected to remove all of them. Insufficient removal of axons or axon regeneration may have caused the weak regeneration in the denervated mandible sample on the day 45 (Fig. 7B). The presence of ...
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... Our study revealed the presence of axons in teeth in the axolotl mandible. Axons were observed in the pre-invagination oral epithelium (Fig. 5E,F), the developing tooth buds (Fig. 2I,J), and the mature tooth (Fig. 2K). The axons originate from the trigeminal ganglia (Fig. 5A-C), then from many branches in the proximal region of the dentary (Fig. 5A-C). Our denervation procedure targeted major routes but was not expected to remove all of them. Insufficient removal of axons or axon regeneration may have caused the weak regeneration in the denervated mandible sample on the day 45 (Fig. 7B). The presence of nerves is necessary for successful tooth regeneration and replacement in ...
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... that, in the denervated mandible, invagination of the oral epithelium was delayed and Shh expression in the pre-invagination oral epithelium was suppressed (Figs. 6F and 9D,G), show that nerves play a role in the tooth bud induction process. Furthermore, axon presence in the pre-invagination oral epithelium follows a very suggestive pattern (Fig. 5E' ,F'). Axon fibers were more densely concentrated in the regions adjacent to the invaginating dental lamina, where the next dental lamina were about to emerge. This suggests that nerves play a role in dental lamina invagination. In keeping with hypothesis, the number of newly formed tooth buds was severely decreased in the denervated ...
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... suggest that the dental lamina come from the residual oral epithelium. The present study, however, demonstrates the importance of nerve presence for tooth regeneration in the axolotl mandible. Therefore, it is possible that the results of the previous studies arose in part from unintentional nerve damage caused by the procedures. As shown in Fig. 5A-C, the nerves in this region come from the caudal direction. In Graver's study 14 demonstrating that proximal removal of the dentary resulted in a toothless structure, the removal of the caudal (proximal) part of the dentine would have resulted in severe loss of nerves as well. In our study, nerve loss prevented tooth regeneration in ...
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... mandibular region. A part of each animal's mandible including the oral epithelium was dissected using forceps and scissors. About half of the dentary was dissected out, while the Meckel's cartilage was left intact. In cases of denervation, axon dissection was performed on the same day as dentectomy. The first denervation points are indicated in Fig. 5B. The second denervation was performed on day 10; the dissection points used in this denervation are shown in Fig. 5A. After each surgery, animals were kept on ice for 2 hours to allow their wounds to heal. All animals were subsequently kept in ...
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... About half of the dentary was dissected out, while the Meckel's cartilage was left intact. In cases of denervation, axon dissection was performed on the same day as dentectomy. The first denervation points are indicated in Fig. 5B. The second denervation was performed on day 10; the dissection points used in this denervation are shown in Fig. 5A. After each surgery, animals were kept on ice for 2 hours to allow their wounds to heal. All animals were subsequently kept in ...
Citations
... This method collects serial block-face images of a frozen specimen using a consumer-grade, digital single-lens reflex camera and a standard cryostat and reconstructs a 3D image of the specimen by stacking the collected images in a computer. The method effectively works with naturally colored or histochemically stained, relatively large specimens, such as whole mouse embryos and juvenile zebrafish stained with hematoxylin or tannic acid, and does not require expensive, specialized fluorescence microscopes (Ishii et al., 2021;Makanae et al., 2020;Sutrisno et al., 2023). To visualize target tissues for 3D imaging analysis with the CoMBI method, we chose to use a heat-stable, chromogenic reporter, human placental alkaline phosphatase (generally referred to as PLAP; the official symbol is ALPP) (Deprimo et al., 1996;Fekete & Cepko, 1993), in place of green fluorescent protein (GFP), which has been widely used in Xenopus transgenesis (Ogino & Ochi, 2009). ...
Xenopus is one of the essential model systems for studying vertebrate development. However, one drawback of this system is that, because of the opacity of Xenopus embryos, 3D imaging analysis is limited to surface structures, explant cultures, and post‐embryonic tadpoles. To develop a technique for 3D tissue/organ imaging in whole Xenopus embryos, we identified optimal conditions for using placental alkaline phosphatase (PLAP) as a transgenic reporter and applied it to the correlative light microscopy and block‐face imaging (CoMBI) method for visualization of PLAP‐expressing tissues/organs. In embryos whose endogenous alkaline phosphatase activities were heat‐inactivated, PLAP staining visualized various tissue‐specific enhancer/promoter activities in a manner consistent with green fluorescent protein (GFP) fluorescence. Furthermore, PLAP staining appeared to be more sensitive than GFP fluorescence as a reporter, and the resulting expression patterns were not mosaic, in striking contrast to the mosaic staining pattern of β‐galactosidase expressed from the lacZ gene that was introduced by the same transgenesis method. Owing to efficient penetration of alkaline phosphatase substrates, PLAP activity was detected in deep tissues, such as the developing brain, spinal cord, heart, and somites, by whole‐mount staining. The stained embryos were analyzed by the CoMBI method, resulting in the digital reconstruction of 3D images of the PLAP‐expressing tissues. These results demonstrate the efficacy of the PLAP reporter system for detecting enhancer/promoter activities driving deep tissue expression and its combination with the CoMBI method as a powerful approach for 3D digital imaging analysis of specific tissue/organ structures in Xenopus embryos.
... Nerves are key to understanding amphibian regeneration ability. Nerve presence is essential for limb, tail, tooth, and gill regeneration (Endo et al., 2004;Maden and Holder, 1984;Makanae et al., 2016bMakanae et al., , 2020Saito et al., 2019;Singer, 1952). Our previous study showed that axolotl skin could regenerate its lattice-patterned collagen structure under the presence of nerves after skin removal (Kashimoto et al., 2022). ...
... Due to the great influence of the nerves in successful organ regeneration in amphibians, nerve factors have been sought for a long time. Our previous studies identified FGF2, FGF8, and BMP2 as the nerve factors that can induce multiple organ regeneration in axolotls (Makanae et al., 2014(Makanae et al., , 2016b(Makanae et al., , 2020Saito et al., 2019;Satoh et al., 2016). Among them, FGFs play an essential role in the induction of limb regeneration (Makanae et al., 2014;Mullen et al., 1996). ...
... Among them, FGFs play an essential role in the induction of limb regeneration (Makanae et al., 2014;Mullen et al., 1996). While organ regeneration is completely inhibited in aneurogenic conditions, application of FGFs can trigger organ regeneration processes in denervated limbs, tail, gills, and teeth (Makanae et al., 2014(Makanae et al., , 2016b(Makanae et al., , 2020Saito et al., 2019). However, whether FGFs also induce skin regeneration has not yet been confirmed. ...
Axolotls have been considered to be able to regenerate their skin completely. Our recent study updated this theory with the finding that the lattice structure of dermal collagen fibers was not fully regenerated after skin injury. We also discovered that nerves induce the regeneration of collagen fibers. The mechanism of collagen fiber regeneration remains unknown, however. In this study, we focused on the structure of collagen fibers with collagen braiding cells, and cell origin in axolotl skin regeneration. In the wounded dermis, cells involved in skin repair/regeneration were derived from both the surrounding dermis and the subcutaneous tissue. Regardless of cell origin, cells acquired the proper cell morphology to braid collagen fiber with nerve presence. We also found that FGF signaling could substitute for the nerve roles in the conversion of subcutaneous fibroblasts to lattice-shaped dermal fibroblasts. Our findings contribute to the elucidation of the fundamental mechanisms of true skin regeneration and provide useful insights for pioneering new skin treatments.
... For example, dentectomy studies show that tooth regeneration is a nerve-dependent process while the lower jaw can regenerate without innervation. 103 Also, axolotls can only regenerate their lens within the first 2 weeks of life, contrasting with other tissues' extended regenerative capacity. 79 Apoptotic tissue degradation is a precursor to the regenerative process following axolotl limb injuries, and an axolotl must remove injured cells and reduce immune cell counts to a specific balance to avoid unwanted damage. ...
Ambystoma mexicanum (axolotl) embryos and juveniles have been used as model organisms for developmental and regenerative research for many years. This neotenic aquatic species maintains the unique capability to regenerate most, if not all, of its tissues well into adulthood. With large externally developing embryos, axolotls were one of the original model species for developmental biology. However, increased access to, and use of, organisms with sequenced and annotated genomes, such as Xenopus laevis and tropicalis and Danio rerio, reduced the prevalence of axolotls as models in embryogenesis studies. Recent sequencing of the large axolotl genome opens up new possibilities for defining the recipes that drive the formation and regeneration of tissues like the limbs and spinal cord. However, to decode the large A. mexicanum genome will take a herculean effort, community resources, and the development of novel techniques. Here, we provide an updated axolotl‐staging chart ranging from one‐cell stage to immature adult, paired with a perspective on both historical and current axolotl research that spans from their use in early studies of development to the recent cutting‐edge research, employment of transgenesis, high‐resolution imaging, and study of mechanisms deployed in regeneration.
... Salamanders are a powerful model to study regeneration. First, salamanders possess the most expansive regenerative ability among vertebrates, as they regenerate a wide array of tissues and organs (e.g., limbs, spinal cord, tail, retina, brain, heart, ovaries, lung, and teeth) (Carlson 2011, Chernoff et al. 2003Tsonis and Del Rio-Tsonis 2004;Maden, Manwell, and Ormerod 2013;Neff, Dent, and Armstrong, 2004;Erler, Sweeney, Monaghan 2017;Makanae et al. 2020, Jensen et al. 2021. Second, salamanders maintain the ability to regenerate limbs and tail throughout development and adulthood, even though rate of regeneration slows down as a function of age ). ...
Among vertebrates, salamanders are champion regenerators, as they are able to regenerate a diverse set of tissues, and regenerate throughout their life. While it was originally thought that all salamanders regenerate similarly, recent studies comparing limb regeneration among deeply diverged salamanders (e.g. the axolotl and newts, ~150 million years) have identified deeply conserved commonalities, clear cellular and genomic deviations in the regeneration process. However, as salamanders belong to a speciose and morphologically diverse group, it is possible that cellular and transcriptional differences might also be present among closely related salamanders. Here, I performed a comprehensive transcriptomic analysis to rigorously identify transcriptional similarities and differences among three salamander species of varying divergences: A. mexicanum, A. andersoni, and A. maculatum. Most of these genes were associated with key regeneration processes noted in previous studies, suggesting that they are critical for the first 24 hours of limb regeneration. Unexpectedly however, genes responsible for tissue histolysis and muscle specific genes showed deviation in their regulation, suggesting that the transcriptional program for muscle histolysis varies among species that appear to complete regeneration at the same time. Further analysis of additional time points showed a similar result, suggesting that transcriptional networks deployed during limb regeneration may be evolutionary labile.
In addition, I developed and optimized a bioinformatics pipeline to investigate DNA
methylation organization in the axolotl and better understand how this epigenetic mark is
organized in a large, heavily repetitive, vertebrate genome. I used these methods, as well
as methods to meta-analyze gene expression datasets of axolotl embryo tail regeneration,
to identify significantly expressed regeneration genes that are differentially methylated
during embryo tail regeneration process. The results from these analyses showed that many genes previously implicated as important during limb regeneration also exhibit significant changes in transcription and DNA methylation during embryo tail regeneration. This suggests that changes in gene expression and DNA methylation for these genes are linked and provides a starting point for future research in interrogating DNA methylation states and expression of these genes during regeneration.
... Thus, nerve-secreted factors, which play a role in tail blastema formation, require more investigation. Tooth regeneration is also depending on the presence of nerves (Makanae et al., 2020). Classical studies on tooth regeneration were completed in newts (Davit-Béal et al., 2007;Goss & Stagg, 1958;Goss, 1969). ...
... Tooth regeneration was investigated as an additional phenomenon besides mandibular regeneration. The trigeminal nerves provide axons to axolotl teeth and express Fgf genes in addition to the DRG neurons and the spinal cord (Makanae et al., 2020). Denervation of axons from the trigeminal ganglia resulted in a severe delay in tooth regeneration. ...
... Ectopic expression of Fgf2, Fgf8, and Bmp7 can reverse the delay in tooth regeneration that is caused by denervation. However, denervation could not completely prevent tooth regeneration (Makanae et al., 2020). Denervation procedures in the axolotl regeneration study are usually accompanied by axon regrowth at the end. ...
Amphibians have a very high capacity for regeneration among tetrapods. This superior regeneration capability in amphibians can be observed in limbs, the tail, teeth, external gills, the heart, and some internal organs. The mechanisms underlying the superior organ regeneration capability have been studied for a long time. Limb regeneration has been investigated as the representative phenomenon for organ-level regeneration. In limb regeneration, a prominent difference between regenerative and nonregenerative animals after limb amputation is blastema formation. A regeneration blastema requires the presence of nerves in the stump region. Thus, nerve regulation is responsible for blastema induction, and it has received much attention. Nerve regulation in regeneration has been investigated using the limb regeneration model and newly established alternative experimental model called the accessory limb model. Previous studies have identified some candidate genes that act as neural factors in limb regeneration, and these studies also clarified related events in early limb regeneration. Consistent with the nervous regulation and related events in limb regeneration, similar regeneration mechanisms in other organs have been discovered. This review especially focuses on the role of nerve-mediated fibroblast growth factor in the initiation phase of organ regeneration. Comparison of the initiation mechanisms for regeneration in various amphibian organs allows speculation about a fundamental regenerative process.
Highlights
• 1. Urodeles have a remarkable organ-level regeneration ability.
• 2. Nerve-secreted Fgfs are the key molecules to initiate organ-level regeneration.
• 3. Fibroblast growth factor (FGF)-signaling is important for the initiation of regeneration in multiple organs and species.
... Experiments on tooth regeneration in the salamander, Ambystoma mexicanum, provided further evidence on the indispensable role of the nervous system in tooth bud induction; denervation of the mandible prevented Shh expression and inhibited the invagination of the dental lamina and tooth regeneration. The local innervation performs its ontogenetic function by secreting Fgf and Bmp (Makanae et al., 2020). Schematic diagram of our model of the NVB (neurovascular bundle) niche and the in vivo origin of incisor MSCs. ...
Even though the role of the nervous system in animal development has never been a special object of biological research, surprisingly extensive relevant evidence emanated from studies designed for other purposes. This evidence reveals the unrivaled and unique role of the nervous system in animal development. To the best of my knowledge, the inductive role of the nervous system in organogenesis and histogenesis was first emphasized by the Canadian developmental biologist (Hall, 1998a, b), when he pointed out that the incipient CNS immediately engenders a network of inductions that give rise to different cells, tissues, and organs of embryos and adults (Hall, 1998a, b).
... Experiments on tooth regeneration in the salamander, Ambystoma mexicanum, provided further evidence on the indispensable role of the nervous system in tooth bud induction; denervation of the mandible prevented Shh expression and inhibited the invagination of the dental lamina and tooth regeneration. The local innervation performs its ontogenetic function by secreting Fgf and Bmp (Makanae et al., 2020). Schematic diagram of our model of the NVB (neurovascular bundle) niche and the in vivo origin of incisor MSCs. ...
Even though the role of the nervous system in animal development has never been a special object of biological research, surprisingly extensive relevant evidence emanated from studies designed for other purposes. This evidence reveals the unrivaled and unique role of the nervous system in animal development. To the best of my knowledge, the inductive role of the nervous system in organogenesis and histogenesis was first emphasized by the Canadian developmental biologist (Hall, 1998a, b), when he pointed out that the incipient CNS immediately engenders a network of inductions that give rise to different cells, tissues, and organs of embryos and adults (Hall, 1998a, b).
... This correlation can add molecular data to the 3D morphological data, although the molecular data are limited to 2D distribution. To date, the CoMBI-C system has been used in various research fields, such as 3D analysis of primordia of beetle horn 16 , 3D distribution analysis of myelinated fibers of human facial nerves 17 , 3D morphological analysis of polycystic kidney of knockout mice 18 , and 3D visualization of regenerating bones and cartilage of the axolotl jaw 19 . Through these collaborative studies and ongoing collaborations, we found that users of the conventional CoMBI-C system desired compatibility with not only frozen specimens but also paraffin-embedded specimens. ...
Correlative microscopy and block-face imaging (CoMBI), a method that we previously developed, is characterized by the ability to correlate between serial block-face images as 3-dimensional (3D) datasets and sections as 2-dimensional (2D) microscopic images. CoMBI has been performed for the morphological analyses of various biological specimens, and its use is expanding. However, the conventional CoMBI system utilizes a cryostat, which limits its compatibility to only frozen blocks and the resolution of the block-face image. We developed a new CoMBI system that can be applied to not only frozen blocks but also paraffin blocks, and it has an improved magnification for block-face imaging. The new system, called CoMBI-S, comprises sliding-type sectioning devices and imaging devices, and it conducts block slicing and block-face imaging automatically. Sections can also be collected and processed for microscopy as required. We also developed sample preparation methods for improving the qualities of the block-face images and 3D rendered volumes. We successfully obtained correlative 3D datasets and 2D microscopic images of zebrafish, mice, and fruit flies, which were paraffin-embedded or frozen. In addition, the 3D datasets at the highest magnification could depict a single neuron and bile canaliculus.
Correlative microscopy and block-face imaging (CoMBI) is an imaging method, which is characterized by the ability to obtain both serial block-face images as a 3-dimentional (3D) dataset and sections for 2-dimentional (2D) light microscopic analysis. These 3D and 2D morphological data can be correlated with each other to facilitate data interpretation. CoMBI is an easy-to-install and low-cost 3D imaging method since its system can be assembled by the researcher using a regular microtome, consumer digital camera, and some self-made devices, and its installation and instruction manuals are open-source. After the first release of CoMBI method from our laboratory, CoMBI systems have been installed in more than a dozen laboratories and are used for 3D analysis of various biological specimens. Typical application of CoMBI is 3D anatomical analysis using the natural color and contrast of the specimen. We have been using CoMBI for analyzing human brain to obtain the fine 3D anatomy as a reference to determine the causes of neurological diseases and to improve the effectiveness of surgery. Recently, we have been using CoMBI for detecting the colors of chromogens, which are used for labeling specific molecules. Mouse embryos colored with X-gal, a conventional chromogen for detecting LacZ products, were imaged using CoMBI, and the 3D distribution of X-gal was successfully visualized. Thus, CoMBI can now be used for many purposes, including 3D anatomical analysis, 2D microscopy using sections, and 3D distribution of specific molecules. These suggest that CoMBI should be more widely used in the field of biological research.
Visualization of spatiotemporal expression of a gene of interest is a fundamental technique for analyzing the involvements of genes in organ development. In situ hybridization (ISH) is one of the most popular methods for visualizing gene expression. When conventional ISH is performed on sections or whole‐mount specimens, the gene expression pattern is represented in 2‐dimensional (2D) microscopic images or in the surface view of the specimen. To obtain 3‐dimensional (3D) data of gene expression from conventional ISH, the “serial section method” has traditionally been employed. However, this method requires an extensive amount of time and labor because it requires researchers to collect a tremendous number of sections, label all sections by ISH, and image them before 3D reconstruction. Here, we proposed a rapid and low‐cost 3D imaging method that can create 3D gene expression patterns from conventional ISH‐labeled specimens. Our method consists of a combination of whole‐mount ISH and Correlative Microscopy and Blockface imaging (CoMBI). The whole‐mount ISH‐labeled specimens were sliced using a microtome or cryostat, and all block‐faces were imaged and used to reconstruct 3D images by CoMBI. The 3D data acquired using our method showed sufficient quality to analyze the morphology and gene expression patterns in the developing mouse heart. In addition, 2D microscopic images of the sections can be obtained when needed. Correlating 2D microscopic images and 3D data can help annotate gene expression patterns and understand the anatomy of developing organs. These results indicated that our method can be useful in the field of developmental biology.
