Comparison of T3 responsiveness between larval and adult myogenic systems.
Amphibian metamorphosis provides an excellent model to study remodeling of the body. This phenomenon is characterized by overall remodeling of the body plan (i.e. larval body) which was once established in early embryogenesis. This metamorphic organ remodeling is induced by thyroid hormone (Gudernatsch, 1912; Kaltenbach, 1953) and a larval body is thus converted into an adult one (Ishizuya-Oka and Shi,Y.B, 2007; Miller, 1996). During this period, most of preexisted larval body organs, i.e., ‘larval-specific organs’ such as tail and gills degenerated (Nishikawa and Hayashi, 1995) and new ‘adult-specific organs’ such as fore- and hindlimbs formed (Brown et al., 2005). This cell replacement is thought to be essential for amphibian metamorphosis and deeply involved in various fundamental biological processes such as cell growth, programmed cell death, differentiation, morphogenesis and cell-cell and cell-environment interactions (Nishikawa, 1997; Shibota et.al., 2000; Shimizu-Nishikawa et al., 2002; Yamane et al., 2011). How are these remodeling-events regulated by metamorphic hormones, triiodothyronine (T3) and thyroxine (T4)? It will provide a great value for developmental and endocrinological research to understand the regulatory mechanism. This is because, thyroid hormone work not only for inducing amphibian metamorphosis but also for triggering metamorphosis in the fishes, such as flounder (Miwa and Inui, 1987) and conger eel (Kitajima et al., 1967), and also in sea urchin (Chino et al., 1994). The process of metamorphosis in the anuran is usually coupled with biphasic development (ancestral life-history). However, in species of direct developer such as the Puerto Rican tree frog,
The reason for adopting thyroid hormone as a metamorphosis-inducing trigger by many organisms is involved in the fact that thyroid hormone is a ligand for nuclear receptors (transcription factors) and can directly cause tissue-specific gene expression changes in the same way as steroid hormone work in insect metamorphosis. Thus, in many metamorphosing organisms, their rebuilding from larval to adult body were achieved through switching of gene expression from larval to adult program by regulation of hormonal concentration and spatiotemporal expression of the receptors. For example, in developing limb buds, the expression of thyroid hormone receptor (TR)-β increases at early metamorphic period but down-regulates at metamorphic climax stage. While, in the tail, the TR-β upregulation occurs at metamorphic climax when tail shortening occurs (Yaoita and Brown, 1990).
A key feature of thyroid hormone action during metamorphosis is the multifaceted nature. The hormone promotes some tissue cells to grow and differentiate but induces the other ones to stop proliferation and degenerate (cell death). Although it is obvious that each program (growth or death) is triggered by thyroid hormone action, it still unknown how it causes the different reactions, death or live, only to a particular (larval or adult-type) cells among many TR-β-expressing cells. This is one of the most important issues in developmental biology field. In other word, it is important to understand not only the thyroid hormone actions but also the mechanisms by which some cells are programmed to commit to a specific cell fate (larval or adult type cells). It would be important to focus on cell-to-cell interactions or cell-to-cellular environments for the analysis of regulatory mechanism of cell fates. The environmental factors include nutrients, growth factors, hormones, cytokines, morphogens and extracellular matrices. There are basically two types of the target cells for such factors, i.e. adult and larval type cells. The adult type cells (or adult precursor cells) start their adult gene program by the actions from such factors while the larval type cells respond to the same factors to stop larval program and activate cell death program. In order to clarify this completely different mode of hormonal actions in organ remodeling during metamorphosis, it would be necessary to fully analyze not only hormonal response of target cells but also the interaction between target cells and surrounding cells.
Muscle remodeling also occurs during anuran metamorphosis (Nishikawa and Hayashi, 1994). It is of great interest to study muscle remodeling from larval to adult type during anuran metamorphosis from the aspect of molecular and cellular interactions. This is because, during anuran metamorphosis there are three different muscle changes, 1) degeneration of larval muscle in the tail (Kerr et al., 1974), 2) formation of new muscle (secondary myogenesis) in developing limbs, and 3) conversion of muscles from larval-type to adult-type in the trunk region (Ryke, 1953), which provide a useful system for analyzing programmed muscle cell death and initiation of adult program of myogenesis. In addition to this, it is also important from the viewpoint of evolutionary adaptation of myogenesis in the transition from fish to tetrapod trunk (Glimaldi et al., 2004). Thus, this chapter concentrates on
2. Muscle remodeling during metamorphosis of
In evolutionary history, amphibians are in the process of evolving from aquatic fishes into terrestrial vertebrates. There are many changes in body organs between aquatic and terrestrial species. The most obvious example of such change would be seen in the epidermal changes in the skin. The stratified and keratinized (or cornified) epidermis in whole body skin is one of the phenotypes macroevolutionaly-acquired in amphibians but not fishes and became a well-established characteristic feature of tetrapoda, i.e. terrestrial vertebrates. Since amphibians have an aquatic larval period, the skin of the larva is a fish type of non-cornified epidermis. During metamorphosis, differentiation of a terrestrial type epidermis (i.e. stratified and cornified epidermis) occurs and complete transformation of whole body skin with terrestrial-type cornified (keratinized) epidermis is achieved after metamorphosis. Thus, in the amphibian, it is a unique feature that macroevolutional (phylogenic) changes from fish to terrestrial type are replicated during metamorphic (ontogenic) changes. Other than skin, is there any organs that are converted from larval (or aquatic) to adult (or terrestrial) type during metamorphosis? Just as the skins had evolved so as to protect whole body from drying in a terrestrial environment, it would be needed for the muscles to evolve from aquatic to terrestrial type providing with increased muscle strength so as to overcome the intense gravitational force in the terrestrial environment.
From this point of view, Nishikawa and Hayashi (1994) analyzed electrophoretically the difference in profiles of muscle contractile protein between larval and adult dorsal muscles in the frog
There are two possibilities for the dorsal muscle isoform conversion during metamorphosis (Fig.2A). In the hypothesis 1 (H1), the isoform transition occurs by cell replacement with anterior-posterior proliferation of adult-type myoblasts and death of preexisting larval–type fibers. On the other hands, in hypothesis 2 (H2), the switch in gene expression from larval to adult program occurs within the same cells without larval cell death. If the former (H1) is the case, myoblasts proliferation should occur with anterior-posterior gradient just before the isoform transition in dorsal muscles. Examination of this point by the assay of DNA synthetic activity (Nishikawa and Hayashi, 1994) and PCNA (proliferating cell nuclear antigen) expression (Kawakami et al., 2009) revealed that the cell proliferation activity is higher in the anterior than the posterior dorsal muscles during early metamorphosis just prior to the isoform changes.
These proliferation activities are well-matched the observations that small portions expressing β-TM (adult muscle area) appeared first at dorsomedial (DM) parts of dorsal muscles and the area gradually expanded to overall dorsal muscles (Fig.3 A, B). The DM parts correspond to the “cord” of the tadpole axial muscles which is reported by Elinson et al. (1999). The “cords” in tail portion also express adult muscle isoforms but this (β-TM+) regions never increased during metamorphosis. On the other hands, in β-TM (-) regions, i.e larval muscle areas, many apoptotic dying muscle cells were observed (Fig.3 B, C). From these results, it has been found that the larval to adult muscle remodeling is achieved by cell replacement (H1: “cell replacement model”) with new proliferation of adult myoblasts and death of preexisting larval cells, not by changing gene expression program from larval to adult one within the same cells (H2) (Nishikawa and Hayashi, 1994).
In summary, we can understand the muscle remodeling in
3. Mechanism of programmed muscle cell death and macrophage phagocytosis
Death of regenerating tails in anuran amphibians has attracted interests of many researchers and Kerr et al. are not exceptional, who define the specific term “apoptosis” for the processes of non-accidental and active cell death (Kerr et al., 1974). They observed the tail cell death in
Oligonucleosomal DNA fragmentation is thought to be a good biochemical evidence of the apoptotic cells (Kaufmann et al., 2000). Nishikawa and Hayashi (1995) detected electrophoretically the nucleosomal DNA fragmentation in
These are four cellular components, i.e. two types of myogenic stem cells (larval and adult myoblasts) and two types of muscle fibers (adult and larval muscles), and these are responsible primarily for the muscle conversion during
Another feature of the apoptotic processes is a phagocytosis by macrophages of the dying apoptotic bodies. In regenerating tail of
On the other hands, researchers in French (Demeneix’ group) have been greatly contributed to the finding of cell death regulators for the muscle cell death during metamorphosis. The method was developed by de Luze et al. (1993) to introduce genes directly into tail muscles and using this method Sachs et al. (1997) found that somatic gene transfer with a mouse
We can see from the above that cell-death signaling in the tail muscle of metamorphosing
4. In vitro characters of two types of myogenic stem cells from
Xenopus laevis: Differential hormonal responses in cell division, cell differentiation and programmed cell death
Are the deaths of larval muscle fibers and myoblasts induced by the thyroid hormone during anuran amphibian metamorphosis? How are the cell division and differentiation for the adult muscle stem cells (i. e. adult myoblasts) regulated by thyroid hormone? To answer these questions, Shibota et al. (2000) established primary cell culture methods for adult- and larval-type myoblasts in the frog,
It was found that there were several significant differences (1-7) in the nature of isolated cells between larval and adult-type myogenic stem cells as described below. (1) The cell size just after isolation in the larval-type (15 μm) was larger than that in the adult-type (5 μm). The size of spreading cells 1 day after inoculation was also larger in the larval type cells (30-50 μm) than in the adult type cells (5-10 μm) (Fig.5A, e and a). (2) Both types of cells could adhere to the plastic culture dish with different adhesion ratios (larval type=30-50%; adult type=50-60%). Most of attached cells (88% larval and 81% adult cells) were desmin-positive, showing the isolated cells to be myoblast-rich populations. (3) The timings of start of myotube-differentiation were quite different between larval and adult types: Myotube-formation by myoblasts fusion started on day 2 or 3 in larval but on day 4 in adult cell cultures (Fig.5A, f and c). (4) There was large difference in growth activity between larval and adult cells: The larval myoblasts increased only 2.5-fold over 6 days of culture but the adult ones 5.5-fold (Fig.5 B). (5) The cultured larval myoblasts responded to the metamorphic hormone, T3, with decrease in the DNA synthetic activity (50%) (Fig.5 F). As the result, T3 decreased the cell numbers (sum of myotubes and myoblasts) in larval cell cultures to 56% of those of control cultures over 6 days (Fig.5 E). On the other hands, T3 did not have much influence on the total cell numbers in adult cultures (Fig.5 D). (6) T3 promoted dose-dependently the differentiation of adult myoblasts into myotubes but diminished that of larval cells by half (Fig.6 A and B). (7) Death of differentiated myotubes was promoted by T3 specifically in larval but not in adult cultures (Fig.5 C and Fig.6 C). In addition to the myotubes death, double staining with TUNEL and anti-desmin (a myoblast marker) antibody showed that death of myoblasts (desmin+ cells) was induced by T3 specifically in larval but not adult cells (Fig.6 D). From the differences in cell sizes, the start-timing of differentiation and cell growth activity between larval and adult myogenic stem cells (1, 3 and 4), it is conceivable that adult myoblast have a more stemness phenotype than the larval myoblasts. This is to say, adult myoblasts cannot enter the myotube-forming stage without dividing many times, but in contrast, the larval myoblasts can immediately go into myotube-differentiation. From this point of view, it would be an important issue to examine differences in gene expression of early myogenic transcription factors, such as
Another essential difference (5-7) between larval and adult cells was found to be the difference in T3 responses (Table 1). It was thus evident that the conversion of a larval to adult myogenic system during metamorphosis becomes possible through totally specific control of cell division, cell differentiation and programmed cell death at a precursor cell level by T3. In studies using with a myoblast cell line (Yaoita and Nakajima, 1997) cloned from
As noted above, there were shown to be essential differences between larval and adult type myoblasts each isolated from tadpole tail and leg muscles. Also in the trunk dorsal muscles during metamorphosis, there should be two different (larval and adult) myogenic stem cells with a life-or-death fate. Do these cells really exist within trunk dorsal muscles? If this is the case, is there a possibility that they form heterokaryon myotubes (“chimeric fate” myotubes) with different two types of cells other than myotubes consisting only of larval or adult cells? In such chimeric myotubes, which fates, a death-fate or a life-fate, possibly be selected in response to T3? In the next section, the study about cell interaction mechanism which regulates the myotubes fate (life-or-death) in the dorsal muscle during metamorphosis of
↓: suppression; ↑: induction or promotion; n.e.: no effect. In the presence of T3, larval type myogenic system of
5. Interaction between larval- and adult-type myogenic precursor cells during metamorphosis: Regulation of cell death fate and adult muscle differentiation
5.1. Differential distribution of larval and adult myoblasts
It was expected that there should be two types of myoblasts (larval- and adult-type muscle stem cells) in trunk dorsal muscles because both larval and adult-type muscles coexist within the same regions. As a first indirect approach for proving the real existence of different two types of myogenic precursor cells in the same trunk muscle, Shimizu-Nishikawa et al. (2002) compared the enhancement of cell death activity in response to T3 among three parts of the muscles, i.e., tadpole trunk, tail and limb muscles, each of which has a different muscle fate (Fig.7 A).
The results showed that the TUNEL+ dying myoblasts were induced by T3 strongly in the tail cells (10-fold induction; from 1.3% to 13%) and moderately in the trunk cells (2-fold; 2.5% to 5%), but not in the limb cells (1% to 1%). The value of cell death induction in trunk cells was between those of tail and limb cells, suggesting the possibility that two types of myogenic stem cells (i.e. T3-inducible and non-inducible cells) are mixed in the trunk muscles. As the second approach, for further direct evidence, isolation of two types (larval and adult) of myoblasts from the trunk dorsal muscles was tried on the basis of their physical natures. The cells dissociated with enzyme-digestion from tadpole trunk dorsal muscles were pre-cultured for three days in high-serum (growth promoting) medium, harvested with trypsin-digestion and used for two-steps cell isolation with a percoll-density gradient centrifugation (a buoyant density-sensitive method) and an albumin-unit gravity sedimentation (a size-sensitive method)(Fig.7 B). The results showed that two different cell types, large (Lg) and small (Sm) cells, were isolated from dorsal muscle at 1:1 (Sm : Lg) ratio. For comparison, the result from the fractionation of the tail muscle cells with the same procedures showed that the two types of cells (Sm and Lg) were also obtained from the tail muscle but their ratio was 5 : 1 (Fig.7 C). Cultivation of these cells (Sm and Lg) revealed that Lg-cells could grow rapidly and T3 decreased the number of Sm-cells but not that of Lg-cells (Fig.7 D). These results suggest that Lg-cells are the adult myoblasts and the Sm-cells are
larval myoblasts. Thus, it was shown that the trunk dorsal muscles contain almost the same number of two different (larval and adult) myoblasts. Interestingly, there were Lg-cells (adult type myoblasts) even in the tail muscle with a small ratio (1/5 of that in the dorsal muscle). In other words, there are many adult type muscle stem (AMS) cells with high growth activity in the dorsomedial (DM) portions of the trunk dorsal muscle but, on the other hands, a small number of AMS cells in the tail DM portions. These results suggest that the life-or-death fates of trunk and tail muscles are determined primarily by the differential distribution of adult myoblasts within the muscles. On the other hand, from the fact that tail muscle does not convert to adult type even though they contain AMS cells, it is conceivable that during metamorphosis the growth and differentiation of AMS cells is specifically activated in the trunk DM portions but suppressed in the tail DM portions. As to this spatial control, Yamane et al. (2011) suggested the possibility that the trunk-specific adult myogenesis is regulated by two cell-interactive mechanisms: a promotion by spinal cord (SP) cells and a suppression by notochord (Nc) cells (see section 6).
5.2. Interaction between adult and larval myoblasts
It is well known that myoblasts at first proliferate, then stop cell divisions and finally fuse among themselves to form multinuclear myotubes toward terminal differentiation. Unlike this, however, there is an exceptional uninuclear myotube formation without myoblast fusion during somitic myogenesis in
In the trunk dorsal muscles of
Then, is there a possibility that the larval muscle fibers in tadpole are rescued from their T3-mediated death by fusing with the adult type myoblasts and transform into adult muscle fibers? If this is the case, such cell-fate conversion may accelerate the speed of adult myogenesis because such tadpoles do not have to use much more energy for destroying a lot of differentiated larval muscles. So, does a fusion-mediated fate-change really occur? For the clarification of this point, it was examined using an in vitro co-culture system whether chimeric myotubes with both adult and larval myoblasts respond to T3 to die or not (Fig.8 B). As the result, it was clarified that both larval and adult myoblasts randomly fuse to each other to make heterokaryon and the rescue from their T3-mediated death occur only when the proportion of adult nuclei number was higher than 80 % within the myotube. Since the rescue from larval cell death thus requires incorporation of so many adult cells, the rescue of the trunk myotubes would occur at a very low rate and most of larval type cells would usually die during metamorphosis by the action of T3. Interestingly, an apoptotic feature (DNA fragmentation) was not observed in any larval nuclei within the surviving heterokaryon myotubes (i.e. adult nuclei ratio ≥ 80%). This mean that the larval nuclei were protected from apoptotic death and their death fate was converted to a life fate (Fig.8 C). However, because a lot of adult cell fusion are needed for preexisting larval muscles to increase the adult-nuclear ratio up to 80%, it is reasonable in vivo situation that adult dorsal muscle conversion by the rescue of the larval myotubes seldom occurs or it occurs only at very few fibers in the anterior portion of body axis with high growth activity. Accordingly, it would never occur in the tail portion where the growth activity of myoblasts is very low (Fig.9). In essence, adult conversion of the trunk dorsal muscles is mainly carried out by the new myotubes formation rather than the old myotubes rescue.
Then, not involving the rescue mechanism, another mechanism which promotes the adult myoblasts differentiation should be needed in order to make efficiently the adult muscles in dorsal muscle region. In order to know whether such promotion of adult myogenesis involves some kinds of cell-cell interactions, experiments with “separated co-culture” of two types of (adult and larval) myoblasts were conducted (Shimizu-Nishikawa et al., 2002). In this experiment, adult (frog leg muscle) and larval (tadpole tail muscle) type myoblasts were separately inoculated in the two different areas in the same culture dish in order to avoid a direct adult-to-larval cell interaction (“separated co-culture”) and their differentiation activity was compared with that in control cultures with either one of the two types by counting myotube and myotube-nuclei numbers within each areas. In this “separated co-culture” system, two types of myoblasts can communicate only through culture medium but through direct cell-to-cell interactions. The result clearly showed that differentiation of adult myoblasts into myotubes was promoted by larval myoblasts but that of larval myoblasts was not affected by adult cells (Fig.10 A).
This effect should be caused by some humoral factors which released from larval myoblasts but not by a direct cell-to-cell contact, because it occurred at a certain distance in the “separated” areas. So, it was examined whether the activity which promotes adult differentiation was observed in conditioned medium (CM). The result indicated that the activity was found only in a larval myoblast CM but not in an adult myoblast CM, suggesting that larval cell secreted a factor(s) for adult muscle differentiation. This putative factor was found to be in the retentate (R) fraction with molecular weight (MW) more than 10,000 through ultra filtration (MW 10,000 cut-off). The ultra filtration also revealed the inverse activity that inhibits the adult myoblast differentiation in the R fraction of control culture medium, suggesting that control medium (maybe serum components in medium) intrinsically contains a factor(s) which antagonizes with the factor(s) in L-CM to inhibit the adult myoblast differentiation. Thus, in order to examine if such inhibitory factor is from serum components of the culture medium, each adult and larval myoblasts were cultured in various conditions with different fetal calf serum (FCS) concentrations and their differentiation (myotube formation) activities were measured (Fig.10 B). As a result, differentiation of adult myoblasts was found to be suppressed dose-dependently by FCS but that of larval cells not to be affected. Taken from these results, it is conceivable that adult differentiation promoting factor(s) being released from larval cells functions through antagonistic regulation of the adult differentiation-inhibitory factor(s) in the control medium (i.e. serum). It was suggested from a work with mouse myogenic cells (Cusella-DeAngelis et al., 1994) that the differentiation inhibitory factor(s) in serum could possibly be some kind of molecules related to TGF-β. Because, interestingly, TGF-β dose-dependently suppressed the differentiation of mouse fetal (but not embryonic) myoblasts in the same way as FCS dose-dependently suppressed that of
In summary, it was found for the first time that the
6. Interaction between adult myogenic precursor cells and axial cells
The adult muscle differentiation occurs in the trunk (but not the tail) during
Therefore, it is reasonable to hypothesize for the trunk-specific adult muscle differentiation that adult myoblast differentiation is promoted by the spinal cord but suppressed by the notochord. So, according to this hypothesis, each of adult (from hindlimb) and larval (from tail) myoblasts was co-cultured with the spinal cord (or notochord) cells so as to compare their responses to two axial cells (i.e. spinal cord and notochord cells)(Fig.12). The result clarified the expected opposite roles of the two axial cells: The spinal cord cells increased twice the myotubes-forming activity of adult myoblasts but did not increase that of larval cells. On the other hands, the notochord cells strongly suppressed the myotube-formation by adult myoblasts but did not suppress that by larval cells (Fig.12 G-L). Thus, there is a high possibility that two contrasting mechanisms, i.e. the “spinal cord (SC)-promotion” and the “notochord (Nc)-suppression” on adult myogenesis, are involved in the trunk-specific adult muscle conversion (Fig.13).
As to the former mechanism, i.e. the “SC-promotion”, the involvement of sonic hedgehog (Shh) signaling is expected, because the spinal cord and notochord expresses Shh and positively regulates the early embryonic myogenesis (Munsterberg and Lassar, 1995, Stern and Hauschka, 1995, Blagden et al., 1997). In fact, the analysis with antibody staining revealed that the N-terminal fragment (active form) of Shh proteins is present in the spinal cord and notochord regions throughout metamorphosis of
On the other hands, as to the “Nc suppression”, the causing factor(s) remains to be unknown. However, another interesting feature of the notochord cells was found by a “separated co-culture” experiment. In the “separated co-culture”, when the notochord cells and adult myoblasts were placed each in separated two areas on the same culture dish in order to avoid direct cell-cell interaction between them, the notochord cells lost their ability to suppress adult myogenesis but rather promoted the adult myoblast differentiation (Fig.16). Interestingly, the same effect (promotion of adult myogenesis) was also observed in a “separated co-culture” with a whole notochord tissue instead of isolated notochord cells. Thus, it was found that notochord cells have a long-distance promotive effect for adult myogenesis (i.e. the “notochord promotion”) other than the “notochord suppression” effect appearing at a short distance. Since the notochord also expresses Shh throughout metamorphosis (Fig.14) and Shh is also known to positively regulate early embryonic myogenesis (Munsterberg and Lassar, 1995), this molecule is possibly be a major candidate molecule for the “notochord promotion”.
As described above, the multiple cell-to-cell interactions coordinately regulate in diverse ways the trunk-specific promotion and the tail-specific suppression of adult myogenesis during metamorphosis. The molecular features of such cell interactions have not fully characterized. Especially, it should be primarily emphasized as an important future work to get insight into the molecular mechanism for the “notochord suppression”. Hebrok et al. (1998) reported with chick embryo that factors from notochord, such as fibroblast growth factors (FGF) and activin, suppress the prepancreatic dorsal endoderm Shh expression and thereby permit early pancreatic development. Such a FGF (or activin)-like molecule(s) might be a factor(s) responsible for the “notochord suppression”. Further investigations are needed for clarifying this question.
Secondary, it is also very important issue to clarify the signaling cascade for the “SC promotion” of adult myogenesis. The “SC promotion” shows adult-specific effect on the muscle differentiation and Shh is expected to be a responsible factor for this phenomenon. As detailed above (section 4), adult myoblasts proliferate many times as undifferentiated stem cells and then after few days stop cell divisions to transit to the differentiation steps (Shibota et al., 2000). Therefore, it is reasonable that the “SC promotion” is involved in the transition step from the undifferentiated stemness stage of the adult cells to the more committed differentiation stage. Borycki et al. (1999) reported that Shh, produced by the notochord and floor plate, control epaxial muscle determination through
The clarification of the cell-cell interaction mechanisms and their molecular cascades for the adult muscle differentiation during
Nishikawa and Hayashi firstly examined the larval-to adult-muscle isoform transition and put forward a new model, the “cell replacement model”, that clearly explains the larval-to-adult myogenic conversion during frog metamorphosis. In this model, larval-to-adult conversion of tadpole dorsal muscles was achieved through the cell replacement by both death of larval-type myogenic cells and proliferation and differentiation of adult-type myogenic cells. The death of tadpole trunk dorsal muscles was found to occur through apoptotic processes including nucleosomal DNA-fragmentation, apoptotic body formation and phagocytosis by macrophages.
In subsequent research, larval- and adult-type myogenic precursor cells (myoblasts) were isolated each from
Finally, co-culture system using myogenic cells (larval and adult myoblasts) and non-myogenic axial cells (notochord and spinal cord cells) was developed to examine how adult myogenesis is promoted in the trunk muscle region but suppressed in the tail muscle region through the interactions between myogenic and non-myogenic cell or environmental signals. The results revealed the suppression of adult myogenesis by notochord cells (“notochord suppression”), promotion of adult myogenesis by spinal cord cells (“spinal cord promotion”) and upregulation of adult myogenesis by sonic hedgehog (Shh)-signaling. These results present a model for the region-specific regulatory mechanism of adult myogenesis by cell-cell interactions, i.e., “spinal cord promotion” and “notochord suppression”, during X. laevis metamorphosis.