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ABSTRACT: Heparan sulfate (HS) interacts with numerous growth factors, morphogens, receptors, and extracellular matrix proteins. Disruption of HS synthetic enzymes causes perturbation of growth factor signaling and malformation in vertebrate and invertebrate development. Our previous studies show that the O-sulfation patterns of HS are essential for the specific binding of growth factors to HS chains, and that depletion of O-sulfotransferases results in remarkable developmental defects in Drosophila, zebrafish, chick, and mouse. Here, we show that inhibition of chick HS-6-O-sulfotransferases (HS6ST-1 and HS6ST-2) in the prospective limb region by RNA interference (RNAi) resulted in the truncation of limb buds and reduced Fgf-8 and Fgf-10 expressions in the apical ectodermal ridge and in the underlying mesenchyme, respectively. HS6ST-2 RNAi resulted in a higher frequency of limb truncation and a more marked change in both Fgf-8 and Fgf-10 expressions than that achieved with HS6ST-1 RNAi. HS6ST-1 RNAi and HS6ST-2 RNAi caused a significant but distinct reduction in the levels of different 6-O-sulfation in HS, possibly as a result of their different substrate specificities. Our data support a model where proper levels and patterns of 6-O-sulfation of HS play essential roles in chick limb bud development.
Embryologia 02/2010; 52(2):146-56. · 2.21 Impact Factor
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ABSTRACT: Regenerating limbs of urodele amphibians and Xenopus tadpole are reconstructed along proximal-distal, anterior-posterior (AP), and dorsal-ventral axes. In contrast, a regenerated limb of the Xenopus froglet does not have digits, and only a simple cartilaginous structure referred to as a "spike" is formed. This suggests that repatterning along the AP axis is absent in the froglet blastema. Previous studies have shown that Shh and its target genes are not expressed in the froglet blastema. In this study, we activated Hedgehog signaling in the froglet blastema and found that target genes of Shh were inducible in the mesenchyme of limb blastema. Furthermore, we found that activation of the signaling had effects on blastema cell proliferation and chondrogenesis and resulted in the formation of multiple cartilaginous structures. These findings indicate that activation of signaling that is absent in the froglet blastema is effective for improvement of limb regeneration ability in the Xenopus froglet.
Developmental Dynamics 07/2009; 238(8):1887-96. · 2.54 Impact Factor
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ABSTRACT: An autoradiographic analysis was undertaken to examine the localization of retinoids applied exogenously to chick limb buds. Ion-exchange beads (AGI-X2) containing a tritium-labeled synthetic retinoid, Am80, were implanted to various regions of chick wing buds. This synthetic retinoid is known to induce a duplicated limb pattern as retinoic acid (RA) does. One to 24 hours after the application, wing buds were fixed, sectioned, and prepared for autoradiography. Heavy labeling was observed in the peripheral region of the wing mesoderm, but no gradient along the antero-posterior axis was found.These results suggest that the peripheral region of the limb bud may be important for the morphogenetic function of RA. Tissue-bound retinoids may not form an antero-posterior concentration gradient when retinoids are added to the anterior margin of the chick limb bud.
Embryologia 07/2008; 35(5):593 - 599. · 2.21 Impact Factor
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ABSTRACT: The expression of two regeneration-associated antigens in the blastemas of normal and retinoid-treated regenerating limbs of axolotl (Ambystoma mexicanum) was examined.One antigen, 55C12, which was similar to tenascin in expression pattern and molecular weight profile, was weakly expressed in the perichondrium and tendon of normal limbs. In the regenerating limbs, the amount of 55C12 antigen increased near the amputation site within 7 days and almost all cells of the blastema mesenchyme came to be positive to the antigen at 20 days, although those of epidermis and most stump tissues were negative. When the regenerating limbs were treated with Am80, a synthetic retinoid, which induced proximo-distal duplication, the expression of 55C12 antigen in the blastema became weak temporarily and was reactivated in the anterior region of the blastema. This expression pattern suggests that the duplicated limb is formed by the preferential growth of this 55C12-positive anterior blastema region.The other antigen, 117C1, was faintly expressed in the epidermis, dermis, muscle, perichondrium and cartilage of normal limbs, and intensely expressed in the blastema mesenchyme and wound epidermis. The Am80 treatment, however, induced no changes in the expression pattern of 117C1.These results suggest that these antigens may distinguish two different regions of the blastema in normal regeneration and retinoid-induced duplication.
Embryologia 07/2008; 36(4):357 - 364. · 2.21 Impact Factor
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ABSTRACT: AV-1 protein is a molecule which shows position-specific expression during chick limb development, and is expected to have some important roles in limb pattern formation. In this study, to examine whether the ZPA (Zone of polarizing activity) effects the expression of the AV-1 protein, we have removed or grafted the ZPA in chick limb buds and observed AV-1 expression. Anterior halves of the limb buds which lack a ZPA were used as hosts. In such anterior halves, AV-1 expression was initially observed in distal mesodermal cells including the cut surface. These anterior halves were combined with ZPA fragments, anterior fragments, posterior half limb buds, or left to develop alone, and the distribution of AV-1 expression was examined. The results of these experiments show that AV-1 expression requires the ZPA, and that expression occurs in the distal mesodermal cells certain distance from the ZPA.
Embryologia 07/2008; 33(2):173 - 180. · 2.21 Impact Factor
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ABSTRACT: The autopod, including the mesopodium and the acropodium, is the most distal part of the tetrapod limb, and developmental mechanisms of autopod formation serve as a model system of pattern formation during development. Cartilage rudiments of the autopod develop after proximal elements have differentiated. The autopod region is marked by a change in the expression of two homeobox genes: future autopod cells are first Hoxa11/Hoxa13-double-positive and then Hoxa13-single-positive. The change in expression of these Hox genes is controlled by upstream mechanisms, including the retinoic acid pathway, and the expression of Hoxa13 is connected to downstream mechanisms, including the autopod-specific cell surface property mediated by molecules, including cadherins and ephrins/Ephs, for cell-to-cell communication and recognition. Comparative analyses of the expression of Hox genes in fish fins and tetrapod limb buds support the notion on the origin of the autopod in vertebrates. This review will focus on the cellular and molecular regulation of the formation of the autopod during development and evolutionary developmental aspects of the origin of the autopod.
Embryologia 05/2008; 50(Supplement s1):S177-S187. · 2.21 Impact Factor
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ABSTRACT: Spermatozoa undergo dramatic physiological changes at fertilization. In the ascidian Ciona intestinalis, an egg-derived substance named SAAF induces both sperm activation and chemotaxis to the egg. To elucidate the molecular mechanism underlying these phenomena, whole sperm proteins before and after SAAF-treatment were analyzed by two-dimensional gel electrophoresis. By comparison of spot patterns before and after activation, we found twelve proteins that changed the isoelectric points. Seven proteins were shown to be axonemal proteins and others were suggested to be non-axonemal components. Analysis of these proteins by MS-based proteomic system revealed that components of several substructures of the axonemes underwent the changes in isoelectric point at sperm activation, including WD-repeat intermediate chains of outer and inner arm dyneins and a radial spoke protein LRR37, as well as novel axonemal proteins with armadillo repeats or SMC domain. Molecules for cell signaling such as 14-3-3 proteins, Skp1 and VCP/p97 also showed isoelectric changes at sperm activation. These results show a comprehensive feature for signaling mechanism of the activation of spermatozoa at fertilization and also shed new lights on the regulation of ciliary and flagellar movements.
Cell Motility and the Cytoskeleton 04/2008; 65(3):249-67. · 4.19 Impact Factor
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ABSTRACT: The Xenopus adult limb has very limited regeneration ability, and only a simple cartilaginous spike structure without digits is formed after limb amputation. We found that expression of Shh and its downstream genes is absent from the regenerating blastema of the Xenopus froglet limb. Moreover, we found that a limb enhancer region of the Shh gene is highly methylated in the froglet, although the sequence is hypomethylated in the Xenopus tadpole, which has complete limb regeneration ability. These findings, together with the fact that the promoter region of Shh is hardly methylated in Xenopus, suggest that regenerative failure (deficiency in repatterning) in the Xenopus adult limb is associated with methylation status of the enhancer region of Shh and that a target-specific epigenetic regulation is involved in gene re-activation for repatterning during the Xenopus limb regeneration process. Because the methylation level of the enhancer region was low in other amphibians that have Shh expression in the blastemas, a low methylation status may be the basic condition under which transcriptional regulation of Shh expression can progress during the limb regeneration process. These findings provide the first evidence for a relationship between epigenetic regulation and pattern formation during organ regeneration in vertebrates.
Developmental Biology 01/2008; 312(1):171-82. · 4.07 Impact Factor
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ABSTRACT: The interactions of heparan sulfate (HS) with heparin-binding growth factors, such as fibroblast growth factors (FGFs), depend greatly on the chain structures. O-Sulfations at various positions on the chain are major factors determining HS structure; therefore, O-sulfation patterns may play a crucial role in controlling the developmental and morphogenetic processes of various tissues and organs by spatiotemporally regulating the activities of heparin-binding growth factors. In a previous study, we found that HS-2-O-sulfotransferase is strongly expressed throughout the mesoderm of chick limb buds during the early stages of development. Here we show that inhibition of HS-2-O-sulfotransferase in the prospective limb region by small inhibitory RNA resulted in the truncation of limb buds and reduced Fgf-8 expression in the apical ectodermal ridge. The treatment also reduced Fgf-10 expression in the mesenchyme. Moreover 2-O-sulfated HS, normally abundant in the basement membranes and mesoderm under ectoderm in limb buds, was significantly reduced in the treated buds. Phosphorylation levels of ERK and Akt were up-regulated in such truncated buds. Thus, we have shown for the first time that 2-O-sulfation of HS is essential for the FGF signaling required for limb bud development and outgrowth.
Journal of Biological Chemistry 08/2007; 282(27):19589-97. · 4.77 Impact Factor
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ABSTRACT: Median fins, unpaired appendages in fish, are fundamental locomotory organs that are believed to have evolved before paired lateral appendages in vertebrates. However, the early process of median fin development remains largely unknown. We investigated the early development of the median fin fold, a rudiment of median fins, and report here the process in zebrafish embryos and the function of FGF signaling in the process. Using expressions of three genes, dlx5a, sp9 and fgf24, as markers of different phases of fold development, our findings suggest that the early process of median fin fold development can be divided into two steps, specification of the median fin fold territory and construction of the fold structure. Both loss-of-function and gain-of-function assays revealed that FGF signaling plays roles in each step, suggesting a common mechanism for the development of median appendages and paired lateral appendages.
Developmental Biology 05/2007; 304(1):355-66. · 4.07 Impact Factor
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ABSTRACT: Understanding the mechanisms that control amphibian limb regeneration should allow us to decipher the critical differences between amphibians and humans, which have the limited ability of organ regeneration. However, many issues at the cellular and molecular levels still remain unresolved. We have generated a transgenic Xenopus laevis line that expresses green fluorescent protein (GFP) under the control of mouse prx1 limb enhancer, which directs reporter gene expression in limb mesenchyme in mice, and found that GFP accumulated in blastemal mesenchymal cells of the transgenic froglets after limb amputation. Thus, this transgenic line should provide a new approach to gain insights into the cellular dynamics and signaling pathways involved in limb blastema formation. We have also developed a culture system for forelimb explants of froglets and found that treatment with inhibitors of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) kinase 1/2 (MEK1/2) and phosphatidylinositol 3-kinase (PI3K) repressed GFP expression. These effects were partially reversible, and down-regulation of GFP was associated with inhibition of cell-cycle progression and induction of ectopic apoptosis. In addition, we found that ERK1/2 and AKT, downstream mediators of MEK1/2 and PI3K pathways, were activated in amputated forelimb stumps. These results demonstrate that MEK/ERK and PI3K/AKT pathways regulate limb blastema formation in the X. laevis froglet.
Developmental Biology 05/2007; 304(2):675-86. · 4.07 Impact Factor
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ABSTRACT: Mammalians have a low potency for limb regeneration compared to that of amphibians. One explanation for the low potency is the deficiency of cells for regenerating amputated limbs in mammals. Amphibians can form a blastema with dedifferentiated cells, but mammals have few such cells. In this paper, we report limb formation, especially bone/cartilage formation in amputated limbs, because bone/cartilage formation is a basic step in limb pattern regeneration. After the amputation of limbs of a neonatal mouse, hypertrophy of the stump bone was observed at the amputation site, which was preceded by cell proliferation and cartilage formation. However, no new elements of bone/cartilage were formed. Thus, we grafted limb buds of mouse embryo into amputated limbs of neonatal mice. When the intact limb bud of a transgenic green fluorescent protein (GFP) mouse was grafted to the limb stump after amputation at the digit joint level, the grafted limb bud grew and differentiated into bone, cartilage and soft tissues, and it formed a segmented pattern that was constituted by bone and cartilage. The skeletal pattern was more complicated when limb buds at advanced stages were used. To examine if the grafted limb bud autonomously develops a limb or interacts with stump tissue to form a limb, the limb bud was dissociated into single cells and reaggregated before grafting. The reaggregated limb bud cells formed similar digit-like bone/cartilage structures. The reaggregated grafts also formed segmented cartilage. When the reaggregates of bone marrow mesenchymal cells were grafted into the stump, these cells formed cartilage, as do limb bud cells. Finally, to examine the potency of new bone formation in the stump tissue without exogenously supplied cells, we grafted gelatin gel containing BMP-7. BMP induced formation of several new bone elements, which was preceded by cartilage formation. The results suggest that the environmental tissues of the stump allow the formation of cartilage and bone at least partially, and that limb formation will be possible by supplying competent cells endogenously or exogenously in the future.
Embryologia 03/2007; 49(2):89-98. · 2.21 Impact Factor
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ABSTRACT: Xenopus has 4 and 5 digits in a forelimb and hindlimb, respectively. It is thought that their limbs and digits develop in Xenopus by mechanisms that are almost conserved from amphibians to higher vertebrates. This is supported by some molecular evidence. The 5'hoxd genes are convenient marker genes for characterizing digits in the chick and mouse. The anteriormost digit is characterized by being hoxd13-positive and hoxd12 (hoxd11)-negative in the chick and mouse. In this study, we revealed that the anteriormost digit of the Xenopus forelimb is hoxd13-positive and hoxd11-positive, that is, a more posterior character than digit I. The order of formation of digit cartilages also suggested that Xenopus forelimb digit identity is II to V, not I to IV. We have also been interested in the relationship between digit identity and shh. The anteriormost digit develops in a shh-independent way. A limb treated with cyclopamine (a shh inhibitor) has a gene expression pattern (hoxd11-negative) similar to that in shh-deficient mice, suggesting that a hindlimb treated with cyclopamine has a digit I character. However, a Xenopus froglet regenerate (spike), which lacks shh expression during its regeneration process, does not have such an expression pattern, being hoxd11-positive. We investigated hoxd11 transcriptions in blastemas that formed in the anteriormost and posteriormost digits, and we found that the blastemas have different hoxd11 expression levels. These findings suggest that the froglet limb blastema does not have a mere digit I character in spite of shh defectiveness and that the froglet limb blastema recognizes its positional differences along the anterior-posterior axis.
Developmental Dynamics 01/2007; 235(12):3316-26. · 2.54 Impact Factor
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ABSTRACT: Using proteomic and immunochemical techniques, we have identified the light and intermediate chains (IC) of outer arm dynein from sperm axonemes of the ascidian Ciona intestinalis. Ciona outer arm dynein contains six light chains (LC) including a leucine-rich repeat protein, Tctex1- and Tctex2-related proteins, a protein similar to Drosophila roadblock and two components related to Chlamydomonas LC8. No LC with thioredoxin domains is included in Ciona outer arm dynein. Among the five ICs in Ciona, three are orthologs of those in sea urchin dynein: two are WD-repeat proteins and the third one, unique to metazoan sperm flagella, contains both thioredoxin and nucleoside diphosphate kinase modules. The remaining two Ciona ICs have extensive coiled coil structure and show sequence similarity to outer arm dynein docking complex protein 2 (DC2) that was first identified in Chlamydomonas flagella. We recently identified a third DC2-like protein with coiled coil structure, Ci-Axp66.0 that is also associated in substoichiometric amounts with Ciona outer arm dynein. In addition, Oda5p, a component of an additional complex required for assembly of outer arm dynein in Chlamydomonas flagella, also groups with this family of DC2-like proteins. Thus, the assembly of outer arm dynein onto doublet microtubules involves multiple coiled-coil proteins related to DC2.
Cell Motility and the Cytoskeleton 11/2006; 63(10):591-603. · 4.19 Impact Factor
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ABSTRACT: Xenopus laevis larvae can regenerate an exact replica of the missing part of a limb after amputation at an early limb bud stage. However, this regenerative capacity gradually decreases during metamorphosis, and a froglet is only able to regenerate hypomorphic cartilage, resulting in a spike-like structure (spike). It has been reported that the spike has tissue deformities, e.g., a muscleless structure. However, our previous study demonstrated that the muscleless feature of the spike can be improved. The existence of other kinds of tissue, such as tendon, has not been clarified. In this study, we focused on the tendon and dermis, and we isolated the scleraxis and dermo-1 genes, which are known to be marker genes for the tendon and dermis, respectively. The expressions of these genes were investigated in both the developmental and regenerating processes of a Xenopus limb. Although muscle was needed to maintain scleraxis expression, scleraxis transcription was detectable in the muscleless spike. Additionally, although grafting of matured skin, including dermal tissue, inhibited limb regeneration, the expression of dermo-1, a dermal marker gene, was detected from the early stage of the froglet blastema. These results indicate that tendon precursor cells and dermal cells exist in the regenerating froglet blastema. Our results support the idea that spike formation in postmetamorphic Xenopus limbs is epimorphic regeneration.
Developmental Dynamics 04/2006; 235(4):1065-73. · 2.54 Impact Factor
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ABSTRACT: Limb regeneration in amphibians is a representative process of epimorphosis. This type of organ regeneration, in which a mass of undifferentiated cells referred to as the "blastema" proliferate to restore the lost part of the amputated organ, is distinct from morphallaxis as observed, for instance, in Hydra, in which rearrangement of pre-existing cells and tissues mainly contribute to regeneration. In contrast to complete limb regeneration in urodele amphibians, limb regeneration in Xenopus, an anuran amphibian, is restricted. In this review of some aspects regarding adult limb regeneration in Xenopus laevis, we suggest that limb regeneration in adult Xenopus, which is pattern/tissue deficient, also represents epimorphosis.
TheScientificWorldJOURNAL 02/2006; 6 Suppl 1:26-37. · 1.66 Impact Factor
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ABSTRACT: In Xenopus laevis, limb buds start to develop at a later point of the larval stage, prior to metamorphosis. This onset of limb development in Xenopus is totally different from that in amniotes such as birds and mammals, in which limb buds emerge at an early stage of embryogenesis, in parallel with other organogenesis. We investigated limb myogenesis in Xenopus, focusing on myogenic gene expression, myogenic ability of limb bud cells in the early stage, and the origin of myogenic precursor cells in the limb bud. The Xenopus early limb bud contains myoD/cardiac actin-positive and pax3/pax7-negative cells. Interestingly, results of transplantation experiments have revealed that this early limb bud contains myogenic precursor cells. In order to know the contribution of myogenic cells in somites to myogenic precursor cells in the early limb bud, we used a Cre-LoxP system for tracing over a long period. The results of fate tracing for myogenic cells in somites of the Xenopus embryo suggested that early-specified myogenic cells in somites do not contribute to limb muscle in Xenopus. Taken together, the results suggest that limb muscle development in Xenopus has characteristics of initiation and early events distinct from those of other vertebrate clades.
Developmental Dynamics 01/2006; 234(4):846-57. · 2.54 Impact Factor
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ABSTRACT: Blastema formation, the initial stage of epimorphic limb regeneration in amphibians, is an essential process to produce regenerates. In our study on nerve dependency of blastema formation, we used forelimb of Xenopus laevis froglets as a system and applied some histological and molecular approaches in order to determine early events during blastema formation. We also investigated the lateral wound healing in comparison to blastema formation in limb regeneration. Our study confirmed at the molecular level that there are nerve-dependent and -independent events during blastema formation after limb amputation, Tbx5 and Prx1, reliable markers of initiation of limb regeneration, that start to be expressed independently of nerve supply, although their expressions cannot be maintained without nerve supply. We also found that cell proliferation activity, cell survival and expression of Fgf8, Fgf10 and Msx1 in the blastema were affected by denervation, suggesting that these events specific for blastema outgrowth are controlled by the nerve supply. Wound healing, which is thought to be categorized into tissue regeneration, shares some nerve-independent events with epimorphic limb regeneration, although the healing process results in simple restoration of wounded tissue. Overall, our results demonstrate that dedifferentiated blastemal cells formed at the initial phase of limb regeneration must enter the nerve-dependent epimorphic phase for further processes, including blastema outgrowth, and that failure of entry results in a simple redifferentiation as tissue regeneration.
Developmental Biology 11/2005; 286(1):361-75. · 4.07 Impact Factor
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ABSTRACT: In Xenopus laevis, amputation of the adult limb results in the formation of a simple (hypomorphic) spike-like structure without joints, although tadpole limb bud regenerates complete limb pattern. The expression of some joint marker genes was examined in limb development and regeneration. Bmp-4 and gdf-5 were expressed and sox-9 expression was decreased in the joint region. Although developing cartilages were well-organized and had bmp-4 expressing perichondrocytes, the spike cartilage did not have such a structure, but only showed sparse bmp-4 expression. Application of BMP4-soaked beads to the spike led to the induction of a joint-like structure. These results suggest that the lack of joints in the spike is due to the deficiency of the accumulation of the cells that express bmp-4. Improvement of regeneration in the Xenopus adult limb that we report here for the first time will give us important insights into epimorphic regeneration.
Developmental Dynamics 09/2005; 233(4):1444-53. · 2.54 Impact Factor
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ABSTRACT: Positioning of the limb is one of the important events for limb development. In the early stage of embryogenesis, the lateral plate mesoderm splits into two layers and the dorsal layer (the somatic mesoderm) gives rise to a series of distinct structures along the rostrocaudal axis, including the forelimb bud, flank body wall, and hindlimb bud. To determine whether positional information in the somatic mesoderm for regionalization along the rostrocaudal axis is also inherited by the ventral layer of the lateral plate mesoderm (the splanchnic mesoderm), experiments in which the splanchnic mesoderm was transplanted under the ectoderm in an in ovo chick system were carried out. Transplantation of the wing-, flank-, and leg-level splanchnic mesoderm resulted in the formation of wings, nothing, and legs, respectively. These results suggest that the splanchnic mesoderm possesses the ability to form limbs and that the ability differs according to the position along the rostrocaudal axis. The position-specific ability to form limbs suggests that there are some domains involved in the formation of position-specific structures in the digestive tract derived from the splanchnic mesoderm, and results of cell fate tracing supported this possibility. In contrast, analysis of shh expression suggested that the anteroposterior polarity in the limb region seems not to be inherited by the splanchnic mesoderm. We propose that the positioning of limb buds is specified and determined in the very early stage of development of the lateral plate mesoderm before splitting and that the polarity in a limb bud is established after the splitting of the mesoderm.
Developmental Dynamics 07/2005; 233(2):256-65. · 2.54 Impact Factor
