FGF signaling regulates mesenchymal differentiation and skeletal patterning along the limb bud proximodistal axis

Fibroblast growth factors (FGFs) are signals from the apical ectodermal ridge (AER) that are essential for limb pattern formation along the proximodistal (PD) axis. However, how patterning along the PD axis is regulated by AER-FGF signals remains controversial. To further explore the molecular mechanism of FGF functions during limb development, we conditionally inactivated fgf receptor 2 (Fgfr2) in the mouse AER to terminate all AER functions; for comparison, we inactivated both Fgfr1 and Fgfr2 in limb mesenchyme to block mesenchymal AER-FGF signaling. We also re-examined published data in which Fgf4 and Fgf8 were inactivated in the AER. We conclude that limb skeletal phenotypes resulting from loss of AER-FGF signals cannot simply be a consequence of excessive mesenchymal cell death, as suggested by previous studies, but also must be a consequence of reduced mesenchymal proliferation and a failure of mesenchymal differentiation, which occur following loss of both Fgf4 and Fgf8. We further conclude that chondrogenic primordia formation, marked by initial Sox9 expression in limb mesenchyme, is an essential component of the PD patterning process and that a key role for AER-FGF signaling is to facilitate SOX9 function and to ensure progressive establishment of chondrogenic primordia along the PD axis.     

Regulation of oligodendrocyte development

Oligodendrocytes are the cells responsible for the formation of myelin in the central nervous system. Recent studies demonstrated that cells of the oligodendrocyte lineage initially arise in distinct regions of the ventricular zone during early development. These cells or their progeny migrate to developing white matter tracts where they undergo the majority of their proliferation and subsequently differentiate into myelinating cells. Oligodendrocyte-precursor cell proliferation is regulated by a number of distinct growth factors that act at distinct stages in the lineage and the final number of oligodendrocytes in any region of the CNS is regulated by local influences. A density-dependent feedback inhibition of proliferation reduces the responsiveness of the cells to their growth factors and the final matching of oligodendrocyte and axon number is accomplished through the local regulation of cell death. In this review, we discuss the major factors that regulate three distinct stages in the development of the oligodendrocyte lineage: The initial induction of oligodendrocyte progenitors, the regulation of expansion and dispersion of the committed precursor cell population, and the final regulation of oligodendrocyte precursor number through the local inhibition of oligodendrocyte precursor proliferation and cell death.     

Wnt and FGF signals interact to coordinate growth with cell fate specification during limb development

A fundamental question in developmental biology is how does an undifferentiated field of cells acquire spatial pattern and undergo coordinated differentiation? The development of the vertebrate limb is an important paradigm for understanding these processes. The skeletal and connective tissues of the developing limb all derive from a population of multipotent progenitor cells located in its distal tip. During limb outgrowth, these progenitors segregate into a chondrogenic lineage, located in the center of the limb bud, and soft connective tissue lineages located in its periphery. We report that the interplay of two families of signaling proteins, fibroblast growth factors (FGFs) and Wnts, coordinate the growth of the multipotent progenitor cells with their simultaneous segregation into these lineages. FGF and Wnt signals act together to synergistically promote proliferation while maintaining the cells in an undifferentiated, multipotent state, but act separately to determine cell lineage specification. Withdrawal of both signals results in cell cycle withdrawal and chondrogenic differentiation. Continued exposure to Wnt, however, maintains proliferation and re-specifies the cells towards the soft connective tissue lineages. We have identified target genes that are synergistically regulated by Wnts and FGFs, and show how these factors actively suppress differentiation and promote growth. Finally, we show how the spatial restriction of Wnt and FGF signals to the limb ectoderm, and to a specialized region of it, the apical ectodermal ridge, controls the distribution of cell behaviors within the growing limb, and guides the proper spatial organization of the differentiating tissues.     

Chondrocytes derived from mouse embryonic stem cells

Our knowledge of cellular differentiation processes during chondro- and osteogenesis, in particular the complex interaction of differentiation factors, is still limited. We used the model system of embryonic stem (ES) cell differentiation in vitro via cellular aggregates, so called embryoid bodies (EBs), to analyze chondrogenic and osteogenic differentiation. ES cells differentiated into chondrocytes and osteocytes throughout a series of developmental stages resembling cellular differentiation events during skeletal development in vivo. A lineage from pluripotent ES cells via mesenchymal, prechondrogenic cells, chondrocytes and hypertrophicchondrocytes up to osteogenic cells was characterized. Furthermore, we found evidence for another osteogenic lineage, bypassing the chondrogenic stage. Together our results suggest that this in vitro system will be helpful to answer so far unacknowledged questions regarding chondrogenic and osteogenic differentiation. For example, we isolated an as yet unknown cDNA fragment from ES cell-derived chondrocytes, which showed a developmentally regulated expression pattern during EB differentiation. Considering ES cell differentiation as an alternative approach for cellular therapy, we used two different methods to obtain pure chondrocyte cultures from the heterogenous EBs. First, members of the transforming growth factor (TGF)-β family were applied and found to modulate chondrogenic differentiation but were not effective enough to produce sufficient amounts of chondrocytes. Second, chondrocytes were isolated from EBs by micro-manipulation. These cells initially showed dedifferentiation into fiboblastoid cells in culture, but later redifferentiated into mature chondrocytes. However, a small amount of chondrocytes isolated from EBs transdifferentiated into other mesenchymal cell types, indicating that chondrocytes derived from ES cells posses a distinct differentiation plasticity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Loss of Eph-receptor expression correlates with loss of cell adhesion and chondrogenic capacity in Hoxa13 mutant limbs.

Mesenchymal patterning is an active process whereby genetic commands coordinate cell adhesion, sorting and condensation, and thereby direct the formation of morphological structures. In mice that lack the Hoxa13 gene, the mesenchymal condensations that form the autopod skeletal elements are poorly resolved, resulting in missing digit, carpal and tarsal elements. In addition, mesenchymal and endothelial cell layers of the umbilical arteries (UAs) are disorganized, resulting in their stenosis and in embryonic death. To further investigate the role of Hoxa13 in these phenotypes, we generated a loss-of-function allele in which the GFP gene was targeted into the Hoxa13 locus. This allele allowed FACS isolation of mesenchymal cells from Hoxa13 heterozygous and mutant homozygous limb buds. Hoxa13(GFP) expressing mesenchymal cells from Hoxa13 mutant homozygous embryos are defective in forming chondrogenic condensations in vitro. Analysis of pro-adhesion molecules in the autopod of Hoxa13 mutants revealed a marked reduction in EphA7 expression in affected digits, as well as in micromass cell cultures prepared from mutant mesenchymal cells. Finally, antibody blocking of the EphA7 extracellular domain severely inhibits the capacity of Hoxa13(GFP) heterozygous cells to condense and form chondrogenic nodules in vitro, which is consistent with the hypothesis that reduction in EphA7 expression affects the capacity of Hoxa13(-/-) mesenchymal cells to form chondrogenic condensations in vivo and in vitro. EphA7 and EphA4 expression were also decreased in the mesenchymal and endothelial cells that form the umbilical arteries in Hoxa13 mutant homozygous embryos. These results suggest that an important role for Hoxa13 during limb and UA development is to regulate genes whose products are required for mesenchymal cell adhesion, sorting and boundary formation.     

Effects of digit tissue on cell death and ectopic cartilage formation in the interdigital zone in chick leg buds

In developing chick leg buds, large-scale cell death occurs in the interdigital zone, which is responsible for the separation of digits from each other. Ectopic cartilage formation is known to occur upon removal of the chondrogenic digit tissue of the leg bud. To examine the mechanisms of ectopic cartilage formation in the interdigital cell death region, we performed the following operations on stage 28–29 leg buds: (i) removal of the digit-forming area; (ii) incision between the interdigital zone and digit region; (iii) insertion of an aluminum barrier into the interdigital zone; and (iv) insertion of a permeable Nuclepore filter into the interdigital zone. In all cases, the inhibition of cell death and/or the formation of ectopic cartilage in the interdigital zone were observed, although the frequency of the inhibition of cell death and the formation of ectopic cartilage varied, depending upon the position where the operations were performed. These results suggest that cell death and cell differentiation in the interdigital zone may be controlled by some factor(s) from digit cartilage.     

Autocrine signals enable chondrocytes to survive in culture

We recently proposed that most mammalian cells other than blastomeres may be programmed to kill themselves unless continuously signaled by other cells not to. Many observations indicate that some mammalian cells are programmed in this way, but is it the case for most mammalian cells? As it is impractical to test all of the hundreds of types of mammalian cells, we have focused on two tissues--lens and cartilage-- which each contain only a single cell type: if there are cells that do not require signals from other cells to avoid programmed cell death (PCD), lens epithelial cells and cartilage cells (chondrocytes) might be expected to be among them. We have previously shown that rat lens epithelial cells can survive in serum-free culture without signals from other cell types but seem to require signals from other lens epithelial cells to survive: without such signals they undergo PCD. We show here that the same is true for rat (and chick) chondrocytes. They can survive for weeks in culture at high cell density in the absence of other cell types, serum, or exogenous proteins or signaling molecules, but they die with the morphological features of apoptosis in these conditions at low cell density. Medium from high density cultures, FCS, or a combination of known growth factors, all support prolonged chondrocyte survival in low density cultures, as long as antioxidants are also present. Moreover, medium from high density chondrocyte cultures promotes the survival of lens epithelial cells in low density cultures and vice versa. Chondrocytes isolated from adult rats behave similarly to those isolated from developing rats. These findings support the hypothesis that most mammalian cells require signals from other cells to avoid PCD, although the signals can sometimes be provided by cells of the same type, at least in tissues that contain only one cell type.     

转录因子对B细胞发育的调控作用

Ｂ细胞同其他血细胞一样,也是由多能造血干细胞发育为祖细胞,再发育成 末成熟细胞的。发育过程中,向Ｂ细胞发育的前 细胞逐渐推动向其他谱系发育的潜能,Ｂ细胞谱系特征性基因开始表达。近来的研究表明,Ｂ细胞发育过程中有多种转录因子参与,它们调节多种重要基因的转录,保证Ｂ细胞的正常发育。本文综述了与Ｂ细胞发育相关转录因子的最新研究进展。     

Wnt/planar cell polarity signaling

The limb is one of the premier models for studying how a simple embryonic anlage develops into complex three-dimensional form. One of the key issues in the limb field has been to determine how the limb becomes patterned along its proximal (shoulder/hip) to distal (digits) axis. For decades it has been known that the apical ectodermal ridge (AER) plays a crucial role in distal outgrowth and patterning of the vertebrate embryonic limb. Most studies have explored the relationship between the AER and the progressive assignment of cell fates to mesenchyme along the proximal to distal (PD) axis. Comparatively few, however, have examined the additional role of the AER to regulate distal outgrowth of the limb and how this growth may also influence pattern along the PD axis. Here, I will review key studies that explore the role of growth in limb development. In particular, I will focus on a recent flurry of papers that examine the role of the Wnt/planar cell polarity (PCP) pathway in regulating directed growth of the limb mesenchyme. Finally, I will discuss a potential mechanism that relates the AER to the Wnt/PCP pathway and how directed growth can play a role in shaping the limb along the PD axis.     

Control of digit formation by activin signalling

Major advances in the genetics of vertebrate limb development have been obtained in recent years. However, the nature of the signals which trigger differentiation of the mesoderm to form the limb skeleton remains elusive. Previously, we have obtained evidence for a role of TGFbeta2 in digit formation. Here, we show that activins A and B and/or AB are also signals involved in digit skeletogenesis. activin betaA gene expression correlates with the initiation of digit chondrogenesis while activin betaB is expressed coincidently with the formation of the last phalanx of each digit. Exogenous administration of activins A, B or AB into the interdigital regions induces the formation of extra digits. follistatin, a natural antagonist of activins, is expressed, under the control of activin, peripherally to the digit chondrogenic aggregates marking the prospective tendinous blastemas. Exogenous application of follistatin blocks physiological and activin-induced digit formation. Evidence for a close interaction between activins and other signalling molecules, such as BMPs and FGFs, operating at the distal tip of the limb at these stages is also provided. Chondrogenesis by activins is mediated by BMPs through the regulation of the BMP receptor bmpR-1b and in turn activin expression is upregulated by BMP signalling. In addition, AER hyperactivity secondary to Wnt3A misexpression or local administration of FGFs, inhibits activin expression. In correlation with the restricted expression of activins in the course of digit formation, neither activin nor follistatin treatment affects the development of the skeletal components of the stylopod or zeugopod indicating that the formation of the limb skeleton is regulated by segment-specific chondrogenic signals.     

Multifaceted control of adult SVZ neurogenesis by the vascular niche

The subventricular zone is one of the 2 germinal niches of the adult brain where neural stem cells (NSC) generate new neurons and glia throughout life. NSC behavior is controlled by the integration of intrinsic signals and extrinsic cues provided by the surrounding microenvironment, or niche. Within the niche, the vasculature has emerged as a critical compartment, to which both neural stem cells and transit-amplifying progenitors are closely associated. A key function of the vasculature is to deliver blood-borne and secreted factors that promote proliferation and lineage progression of committed neural progenitors. We recently found that, in contrast to the established role of soluble cues, juxtacrine signals on vascular endothelial cells maintain neural stem cells in a quiescent and undifferentiated state through direct cell-cell interactions. In this perspective, we discuss how, through these apparently opposing signals, the vascular niche might coordinate stem cell decisions between maintenance and proliferation.     

Strategies to detect interdigital cell death in the frog, Xenopus laevis: T3 accerelation, BMP application, and mesenchymal cell cultivation

Fates of digits in amniotes, i.e., free or webbed digits, are determined by the size of programmed interdigital cell death (ICD) area. However, no (or very few) cell death has thus far been observed in developing limb buds of non-amniotic terrestrial vertebrates including other anuran or urodela amphibians. We speculate that the undetectable situation of amphibian ICD is the result of their less frequency due to slow developmental speed characteristic to most amphibian species. Here, we present three strategies for detecting difficult-to-find ICD in the frog, Xenopus laevis. (1) Addition of triiodo-L-thyronine (T(3)) accelerated two to three times the limb development and increased two to four times the appearance frequency of vital dye-stainable cells in limb buds of the accelerated tadpoles (stage 54 to 55). (2) Application of human bone morphogenetic protein-4 to the autopods of tadpoles at stage 53 to 54 enhanced digital cartilage formation and induced vital dye-stainable cells around the enhanced digital cartilages within 2 d. (3) In cell culture, T(3) increased the chondrogenic and cell death activities of limb mesenchymal cells. The augmentation of both activities by T(3) was stronger in the forelimb cells than in the hindlimb cells. This situation is well coincided with the limb fates of non-webbed forelimbs and webbed hindlimbs in X. laevis adulthood. Collectively, all three approaches showed that it become possible to detect X. laevis ICD with appropriate strategies.     

Apoptosis of Terminally Differentiated Chondrocytes in Culture

During the process of endochondral ossification chondrocytes progress through stages of terminal differentiation culminating in apoptotic death. We have developed a serum-free suspension culture that allows terminal differentiation and facilitates the investigation of factors affecting chondrocyte apoptosis. We have found that chondrocytes not committed to terminal differentiation, i.e., those from the caudal region of chick embryo sterna, a region that remains cartilaginous for some months after the chick hatches, maintained high viability in serum-free suspension culture. A strong dependence of viability on culture density and sensitivity to induction of apoptosis with the protein kinase inhibitor, staurosporine, was consistent with the proposal that these chondrocytes, like nearly all cells, require intercellular communication for survival. Chondrocytes that were committed to terminal differentiation, i.e., those from the cephalic region of chick embryo sterna, a region that is replaced by bone before the chick hatches, expressed the hypertrophic phenotype but maintained their viability in culture for only approximately 6 days. Subsequent cell death was very consistent between cultures and shown to occur by an apoptotic process by analysis of DNA fragmentation and cell morphology. Short-term viability of hypertrophic chondrocytes was independent of culture density and relatively resistant to treatment with staurosporine. Induction of the hypertrophic phenotype in immature chondrocytes committed them to cell death and prevention of expression of the hypertrophic phenotype prevented cell death. We conclude that commitment of chondrocytes to terminal differentiation is associated with a commitment to apoptosis and apoptosis of hypertrophic chondrocytes in growth cartilage does not require initiation by external signals.     

Morphogenesis of the prehensile autopodium in the common chameleon (Chamaeleo Chamaeleo)

This report documents the development of the autopodium of the common chameleon (Chamaeleo chamaeleo) using light microscopy, scanning electron microscopy, and transmission electron microscopy. Three main periods were distinguished during the morphogenesis of this structure. In the first period (stages 33-35 of chameleon development) the autopodium is paddle-shaped with a prominent apical ectodermal ridge (AER) along the distal margin. During this period the AER has structural features similar to other reptilian and avian vertebrates except for the scarcity or absence of gap junctions. The second period of autopodium morphogenesis (stage 36 of chameleon development) is characterized by the formation of a central cleft which divides this structure into two digital segments. In the forelimb the autopodial cleft occupies the space between digits 3 and 4. In the hindlimb the cleft occupies the space between digits 2 and 3. Mesenchymal cell death constitutes a constant feature during cleft formation. In addition to cell death during this process, we have observed that the AER flattens out in the zone of cleft formation while in the digital portions of the autopodium it takes on a polystratified appearance. In the last period of autopodial morphogenesis (stage 37 of chameleon development) digits become free by means of interdigital mesenchymal cell death.     

Osteogenic differentiation of hypertrophic chondrocytes involves asymmetric cell divisions and apoptosis

We have investigated the early cellular events that take place during the change in lineage commitment from hypertrophic chondrocytes to osteoblast-like cells. We have induced this osteogenic differentiation by cutting through the hypertrophic cartilage of embryonic chick femurs and culturing the explants. Immunocytochemical characterization, [3H]thymidine pulse-chase labeling, in situ nick translation or end labeling of DNA breaks were combined with ultrastructural studies to characterize the changing pattern of differentiation. The first responses to the cutting, seen after 2 d, were upregulation of alkaline phosphatase activity, synthesis of type I collagen and single-stranded DNA breaks, probably indicating a metastable state. Associated with the change from chondrogenic to osteogenic commitment was an asymmetric cell division with diverging fates of the two daughter cells, where one daughter cell remained viable and the other one died. The available evidence suggests that the viable daughter cell then divided and generated osteogenic cells, while the other daughter cell died by apoptosis. The results suggest a new concept of how changes in lineage commitment of differentiated cells may occur. The concepts also reconcile previously opposing views of the fate of the hypertrophic chondrocyte.     

Activin/TGFbeta and BMP crosstalk determines digit chondrogenesis

The progress zone (PZ) is a specialized area at the distal margin of the developing limb where mesodermal cells are kept in proliferation and undifferentiated, allowing limb outgrowth. At stages of digit morphogenesis the PZ cells can undergo two possible fates, either aggregate initiating chondrogenic differentiation to configure the digit blastemas, or to die by apoptosis if they are incorporated in the interdigital mesenchyme. While both processes are controlled by bone morphogenetic proteins (BMPs) the molecular basis for such contrasting differential behavior of the autopodial mesoderm remains unknown. Here we show that a well-defined crescent domain of high BMP activity located at the tip of the forming digits, which we termed the digit crescent (DC), directs incorporation and differentiation of the PZ mesenchymal cells into the digit aggregates. The presence of this domain does not correlate with an exclusive expression domain of BMP receptors and its abrogation by surgical approaches or by local application of BMP antagonists is followed by digit truncation and cell death. We further show that establishment of the DC is directed by Activin/TGFβ signaling, which inhibits Smad 6 and Bambi, two specific BMP antagonists expressed in the interdigits and progress zone mesoderm. The interaction between Activin/TGFβ and BMP pathways at the level of DC promotes the expression of the chondrogenic factor SOX9 accompanied by a local decrease in cell proliferation. Characteristically, the DC domain is asymmetric, it being extended towards the posterior interdigit. The presence of the DC is transitorily dependent of the adjacent posterior interdigit and its maintenance requires also the integrity of the AER.     

Regulation of the G1 phase of the mammalian cell cycle

In any multi-cellular organism,the balance between cell division and cell death maintains a constant cell number.Both cell division cycle and cell death are highly regulated events.Whether the cell will proceed through the cycle or not,depends upon whether the conditions required at the checkpoints during the cycle and fulfilled.In higher eucaryotic cells,such as mammalian cells,signals that arrest the cycle usually act at a G1 checkpoint.Cells that pass this restriction point are committed to complete the cycle.Regulation of the G1 phase of the cell cycle is extremely complex and involves many different families of proteins such as retinoblastoma family,cyclin dependent kinases,cyclins,and cyclin kinase inhibitors.     

Smad1/Smad5 signaling in limb ectoderm functions redundantly and is required for interdigital programmed cell death

Bone morphogenetic proteins (BMPs) are secreted signals that regulate apical ectodermal ridge (AER) functions and interdigital programmed cell death (PCD) of developing limb. However the identities of the intracellular mediators of these signals are unknown. To investigate the role of Smad proteins in BMP-regulated AER functions in limb development, we inactivated Smad1 and Smad5 selectively in AER and ventral ectoderm of developing limb, using Smad1 or/and Smad5 floxed alleles and an En1(Cre/+) knock-in allele. Single inactivation of either Smad1 or Smad5 did not result in limb abnormalities. However, the Smad1/Smad5 double mutants exhibited syndactyly due to a reduction in interdigital PCD and an increase in interdigital cell proliferation. Cell tracing experiments in the Smad1/Smad5 double mutants showed that ventral ectoderm became thicker and the descendents of ventral En1(Cre/+) expressing ectodermal cells were located at dorsal interdigital regions. At the molecular level, Fgf8 expression was prolonged in the interdigital ectoderm of embryonic day (E) 13 Smad1/Smad5 double mutants, suggesting that the ectopic Fgf8 expression may serve as a survival signal for interdigital epithelial and mesenchymal cells. Our result suggests that Smad1 and Smad5 are required and function redundantly as intracellular mediators for BMP signaling in the AER and ventral ectoderm. Smad1/Smad5 signaling in the AER and ventral ectoderm regulates interdigital tissue regression of developing limb. Our mutants with defects in interdigital PCD could also serve as a valuable model for investigation of PCD regulation machinery.     

Wnt/planar cell polarity signaling: an important mechanism to coordinate growth and patterning in the limb

The limb is one of the premier models for studying how a simple embryonic anlage develops into complex three-dimensional form. One of the key issues in the limb field has been to determine how the limb becomes patterned along its proximal (shoulder/hip) to distal (digits) axis. For decades it has been known that the apical ectodermal ridge (AER) plays a crucial role in distal outgrowth and patterning of the vertebrate embryonic limb. Most studies have explored the relationship between the AER and the progressive assignment of cell fates to mesenchyme along the proximal to distal (PD) axis. Comparatively few, however, have examined the additional role of the AER to regulate distal outgrowth of the limb and how this growth may also influence pattern along the PD axis. Here, I will review key studies that explore the role of growth in limb development. In particular, I will focus on a recent flurry of papers that examine the role of the Wnt/planar cell polarity (PCP) pathway in regulating directed growth of the limb mesenchyme. Finally, I will discuss a potential mechanism that relates the AER to the Wnt/PCP pathway and how directed growth can play a role in shaping the limb along the PD axis.     

Emergence of chondrogenic progenitor stem cells in transplantation biology-prospects and drawbacks

Avascular tissues such as a cartilage contains a unique type of cell called as the chondrocyte. We, however, have not understood the origin of the chondrocyte population and how this population is maintained in the normal tissue. In spite of being considered to be a simple tissue, scientist had always faced difficulties to engineer this tissue. This is because different structural regions of the articular cartilage were never understood. In addition to this, the limited self-repair potential of cartilage tissue and lack of effective therapeutic options for the treatment of damaged cartilage has remained an unsolved problem. Mesenchymal stem cell based therapy may provide a solution for cartilage regeneration. This is due to their ability to differentiate into chondrogenic lineage when appropriate conditions are provided. An ideal cell source, a three-dimensional cell culture, a suitable scaffold material that accomplishes all the necessary properties and bioactive factors in specific amounts are required to induce chondrocyte differentiation and proliferation. Cartilage tissue engineering is a promising and rapidly expanding area of research that assures cartilage regeneration. However, many unsolved questions concerning the mechanism of engraftment of chondrocytes following transplantation in vivo, biological safety after transplantation and the retention of these cells for lifetime remain to be addressed that is possible only through years of extensive research. Further studies are therefore required to estimate the long-term sustainability of these cells in the native tissue, to identify well suited delivery materials and to have a thorough understanding of the mechanism of interaction between the chondrocytes and extracellular matrix and time is not far when this cell based therapy will provide a comprehensive cure to cartilage disease.