The type I BMP receptor BMPRIB is required for chondrogenesis in the mouse limb

Mice carrying a targeted disruption of BmprIB were generated by homologous recombination in embryonic stem cells. BmprIB(-/-) mice are viable and, in spite of the widespread expression of BMPRIB throughout the developing skeleton, exhibit defects that are largely restricted to the appendicular skeleton. Using molecular markers, we show that the initial formation of the digital rays occurs normally in null mutants, but proliferation of prechondrogenic cells and chondrocyte differentiation in the phalangeal region are markedly reduced. Our results suggest that BMPRIB-mediated signaling is required for cell proliferation after commitment to the chondrogenic lineage. Analyses of BmprIB and Gdf5 single mutants, as well as BmprIB; Gdf5 double mutants suggests that GDF5 is a ligand for BMPRIB in vivo. BmprIB; Bmp7 double mutants were constructed in order to examine whether BMPRIB has overlapping functions with other type I BMP receptors. BmprIB; Bmp7 double mutants exhibit severe appendicular skeletal defects, suggesting that BMPRIB and BMP7 act in distinct, but overlapping pathways. These results also demonstrate that in the absence of BMPRIB, BMP7 plays an essential role in appendicular skeletal development. Therefore, rather than having a unique role, BMPRIB has broadly overlapping functions with other BMP receptors during skeletal development.     

Evidence for regulation following amputation and tissue grafting in the developing mouse limb

Procedures are now available to experimentally manipulate postimplantation mouse embryos in situ and allow development to continue into postnatal life (Muneoka, K., N. Wanek, and S.V. Bryant (1986) J. Exp. Zool., 239:289-293). We have investigated the ability of the well-formed hindlimb bud to regulate following two experimental operations: amputation and wedge grafts designed to confront anterior and posterior cells. After comparing the resultant limbs with the fate maps of the relevant stages, we conclude that the developing hindlimb bud at stage 7/8 (equivalent to stage 27/28 of the chick) is capable of partial regeneration of the peripheral digits following amputation and capable of supernumerary digit tip formation after grafting of a wedge of anterior tissue to a posterior position.     

Arabidopsis desaturase 2 gene is involved in the regulation of cadmium and lead resistance

Aims

It was shown previously that Arabidopsis (Arabidopsis thaliana) desaturase 2 (ADS2) cDNA was isolated and it was shown that the expression of ADS2 was organ-dependent and up-regulated by low temperature. However, little is known about the role of ADS2 gene in heavy metal resistance in plants. In this study, we showed that ADS2 gene is involved in the regulation of cadmium (Cd) and lead (Pb) resistance.

Methods

For heavy metal resistance tests, seeds were germinated and grown on 1/2 MS media supplemented with the indicated concentrations of metal ions. To quantify root length, plants were grown vertically in plates. For heavy metal treatments, two-week old wild-type seedlings grown on MS media were treated with cadmium (Cd) or lead (Pb) for 24 h, and then sampled for metal content measurement and qPCR analysis.

Results

ADS2 was strongly repressed by Cd(II), and ads2-1 mutant plants showed increased Cd(II) resistance. A lower Cd content was detected in ads2-1 plants than in wild-type plants subjected to Cd(II) treatment, which was associated with activation in expression of AtPDR8 gene, a pump excluding Cd(II) and/or Cd(II)-containing toxic compounds from the cytoplasm, suggesting that ADS2-mediated Cd(II) resistance is AtPDR8 dependent. We also found that ads2-1 plants showed increased Pb(II) sensitivity, and ADS2 was strongly repressed by hydrogen peroxide (H₂O₂) but not by Pb(II). The ads2-1 mutant showed increased sensitivity to oxidative stresses mediated by H₂O₂ and paraquat, and higher levels of H₂O₂ accumulation were observed in leaves of ads2-1 plants than those of wild-type plants when subjected to Pb(II) and H₂O₂, indicating that ADS2 mediates Pb(II) resistance indirectly by impaired ROS scavenging.

Conclusions

ADS2 gene mediates Cd(II) and Pb(II) resistance, at least in part, through two distinct mechanisms, an AtPDR8-dependent mechanism and a ROS detoxification system-mediated mechanism, respectively.     

Retinoic acid modulates chondrogenesis in the developing mouse cranial base

The retinoic acid (RA) signaling pathway is known to play important roles during craniofacial development and skeletogenesis. However, the specific mechanism involving RA in cranial base development has not yet been clearly described. This study investigated how RA modulates endochondral bone development of the cranial base by monitoring the RA receptor RARγ, BMP4, and markers of proliferation, programmed cell death, chondrogenesis, and osteogenesis. We first examined the dynamic morphological and molecular changes in the sphenooccipital synchondrosis-forming region in the mouse embryo cranial bases at E12-E16. In vitro organ cultures employing beads soaked in RA and retinoid-signaling inhibitor citral were compared. In the RA study, the sphenooccipital synchondrosis showed reduced cartilage matrix and lower BMP4 expression while hypertrophic chondrocytes were replaced with proliferating chondrocytes. Retardation of chondrocyte hypertrophy was exhibited in citral-treated specimens, while BMP4 expression was slightly increased and programmed cell death was induced within the sphenooccipital synchondrosis. Our results demonstrate that RA modulates chondrocytes to proliferate, differentiate, or undergo programmed cell death during endochondral bone formation in the developing cranial base.     

WIP: a multifunctional protein involved in actin cytoskeleton regulation

Knowledge of the dynamics of actin-based structures is a major key to understanding how cells move and respond to their environment. The ability to reorganize actin filaments in a spatial and temporal manner to integrate extracellular signals is at the core of cell adhesion and cell migration. Several proteins have been described as regulators of actin polymerization: this review will focus on the role of WASP-interacting protein (WIP), an actin-binding protein that participates in actin polymerization regulation and signal transduction. WIP is widely expressed and interacts with Wiskott-Aldrich syndrome protein (WASP) (a hematopoietic-specific protein) and its more widely expressed homologue neural WASP (N-WASP), to regulate WASP/N-WASP function in Arp2/3-mediated actin polymerization. WIP also interacts with profilin, globular and filamentous actin (G- and F-actin, respectively) and stabilizes actin filaments. In vivo WIP participates in filopodia and lamellipodia formation, in T and B lymphocyte activation, in mast cell degranulation and signaling through the Fcepsilon receptor (FcepsilonR), in microbial motility and in Syk protein stability.     

Patterns of chondrogenesis and calcification in the developing limb of the lizard,Calotes versicolor

The first skeletal condensation appears deep at the base of the limb bud near the somites, when the apical ectodermal ridge (AER) is maximally developed. Thereafter the skeletal elements generally appear in a proximodistal sequence but most of the mesopodial cartilages appear well after the metapodial ones and one of them, tarsalia-1, even after the phalangeal ones. The skeletal elements that fuse or “disappear” during the development are the cartilaginous condensation of fibulare, and the precartilaginous condensation of the distal centrale in the tarsus, and possibly the mesenchymatous condensation of the intermedium in the carpus. The calcification of all the long cartilages is perichondral and osseous while that of all the mesopodial and other cartilages, like epiphyses and sesamoids, is endochondral and nonosseous except the partly osseous astragalus and fibulare. The limbs of the mature adult have many sesamoids and metaplastic calcifications. The AER starts regressing after the appearance of the first skeletal condensation but is retained on the digital tips, though in a moderately regressed condition, almost till the time of the appearance of all the phalangeal condensations. These studies on the mesopodium differ with most studies on reptilian and avian mesopodia in favoring the view that very few skeletal condensations fuse or disappear during the development. They thus raise important issues concerning the ontogeny and phylogeny of the pentadactyl limb. While the AER has a substantial role in the limb morphogenesis, it most probably is not responsible for the information to mesoderm regarding the number, size, shape and relative position of the skeletal elements in the limb.     

Shf, a Shb-like adapter protein, is involved in PDGF-alpha-receptor regulation of apoptosis

Recent work has implicated the importance of adapter proteins in signal transduction. To identify homologues of the previously identified adapter protein Shb, database searches were performed. A Shb-like protein was found which we have named Shf. Shf contains an SH2 domain and four putative tyrosine phosphorylation sites and is mainly expressed in skeletal muscle, brain, liver, prostate, testis, ovary, small intestine, and colon. The SH2 domain of Shf bound to the PDGF-alpha-receptor at tyrosine-720, but not to the PDGF-beta-receptor in PAE cells. Pervanadate induced tyrosine phosphorylation of Shf in NIH3T3 fibroblasts overexpressing this protein, whereas PDGF-AA alone had no detectable effect. NIH3T3 cells overexpressing Shf displayed significantly lower rates of apoptosis than control cells in the presence of PDGF-AA. Our findings suggest a role for the novel adapter Shf in PDGF-receptor signaling and regulation of apoptosis.     

Six1 is not involved in limb tendon development, but is expressed in limb connective tissue under Shh regulation

Mice deficient for the homeobox gene Six1 display defects in limb muscles consistent with the Six1 expression in myogenic cells. In addition to its myogenic expression domain, Six1 has been described as being located in digit tendons and as being associated with connective tissue patterning in mouse limbs. With the aim of determining a possible involvement of Six1 in tendon development, we have carefully characterised the non-myogenic expression domain of the Six1 gene in mouse and chick limbs. In contrast to previous reports, we found that this non-myogenic domain is distinct from tendon primordia and from tendons defined by scleraxis expression. The non-myogenic domain of Six1 expression establishes normally in the absence of muscle, in Pax3-/- mutant limbs. Moreover, the expression of scleraxis is not affected in early Six1-/- mutant limbs. We conclude that the expression of the Six1 gene is not related to tendons and that Six1, at least on its own, is not involved in limb tendon formation in vertebrates. Finally, we found that the posterior domain of Six1 in connective tissue is adjacent to that of the secreted factor Sonic hedgehog and that Sonic hedgehog is necessary and sufficient for Six1 expression in posterior limb regions.     

Inhibition of chondrogenesis by normal mouse serum in cultured chick limb cells

Chick limb-bud mesoderm cells from embryonic stages 22–25 were cultured at high cell densities in media known to support chondrogenesis. Under these conditions the continuous presence of normal mouse serum, at a concentration of 10%, inhibits the ability of the cells to produce toluidine blue-stainable cartilage matrix materials. In contrast, mesodermal cells treated with comparable concentrations of other heterologous sera continue to differentiate much like the control cultures while growing in the presence of the test sera. The inhibitory effect of the serum was shown not to be the result of a general cytotoxic effect on protein synthesis or the inability of the cells to incorporate [³H]d-glucosamine. There was a significant difference however, in the distribution of the incorporated glucosamine. Less label was associated with the cell layer of the treated series, while a greater amount of the incorporated material was found to be secreted into the medium when compared with the control cultures. Studies have shown also that the serum inhibitory response is dose dependent, while the factor(s) itself is non-dialysable, stable to heat and repeated freezing and is not a conventional serum lipoprotein. Following the addition of whole or delipidated mouse serum, a significant increase in lipid droplets appears in the cytoplasm of the cells. Biochemical analyses of mouse serum-treated cells indicate that there is a marked increase in their triglyceride content as compared to the control cells. While the nature of the serum inhibitory factor remains to be determined, the accumulation of triglyceride following mouse serum treatment suggests that this may play a role in modulating the expression of the chondrogenic phenotype.     

Patterns of cell polarity in the developing mouse limb

Most cells have a morphological polarity with the centrioles and Golgi apparatus occupying one pole of the cell and the nucleus the other. This structural polarity often correlates with functional polarity as in secretory epithelia where the Golgi apparatus moves to the pole of the cell from which secretory materials are exreted. In limb development an interaction of unknown mechanism occurs between the epithelium and mesenchyme. We have evaluated the pattern of cell polarity using silver impregnation of the Golgi apparatus in limb epithelium and mesenchyme of mouse embryos from day 9.5, when limbs are first visible, to day 15, when cartilage formation is complete. Cells in the epithelium almost always have the Golgi apparatus in the apex of the cell, i.e., oriented away from the basement membrane. The layer of mesenchyme cells just beneath the basement membrane initially has only 16 to 25% of the cells oriented toward the basement membrane. A marked shift in orientation occurs between days 12 and 13 so that from days 13 to 15 up to 53% of the mesenchyme cells are oriented toward the basement membrane. This shift in orientation occurs more slowly in the mesenchyme at a depth of four cells below the basement membrane. This changing pattern of mesenchymal cell polarity occurs at a time when there is an apparent increase in the amount of extracellular matrix, especially in the region just below the basement membrane.     

C-type natriuretic peptide regulation of limb mesenchymal chondrogenesis is accompanied by altered N-cadherin and collagen type X-related functions

AMDM, a form of osteochondrodysplasia, is due to the loss-of-function mutations in NPR-B gene. This study investigated the functional involvement of CNP-3, chick homolog of human CNP, and its receptor NPR-B in chondrogenesis utilizing the micromass culture of the chick limb mesenchymal cells. Results revealed CNP-3 and NPR-B expression in the chick limb bud making stage-specific peak levels first at Hamburger-Hamilton stage 23-24, and second at stage 30-31, corresponding to pre-chondrogenic mesenchymal condensation and initiation of chondrogenic maturation-hypertrophy in vivo, respectively. CNP-3 and NPR-B expression in vitro increased parallel to collagen type X expression, but not to that of collagen type II. Treatment of cultures with CNP significantly increased N-cadherin, and collagen type X expression, glycosaminoglycan synthesis and chondrogenesis. Collagen type II expression was not significantly affected. Thus, results implicated CNP-3/NPR-B signaling in pre-chondrogenic mesenchymal condensation, glycosaminoglycan synthesis and late differentiation of chondrocytes in the process of endochondral ossification.