Initiation of dorso-ventral axis during chick limb development

We analysed spatio-temporal expression of dorso-ventral genes - Wnt-7a, En-1, Lmx-1 and Fgf-8 - during both normal and ectopic limb formation following fibroblast growth factor (FGF) application to the flank. We confirm that Wnt-7a is the first of these genes to be expressed in dorsal ectoderm in limb-forming regions. We also noticed patterns and kinetics of gene expression specific to chick that could account for differences observed in ridge formation between chick and mouse. We find that Wnt-7a expression, in dorsal ectoderm, is rapidly and locally induced by FGF application. In contrast, ectopic induction of Lmx-1 expression, in dorsal mesoderm, is much slower, occurs first at a distance from the FGF-2 bead and seems initially independent of direct Wnt-7a signalling during FGF-2 limb induction. Finally, we show that there is no contribution to extra-limb mesoderm from normal limb mesoderm and confirm that flank cells give rise to the extra limb. Furthermore, we suggest that an inhibitor present in the flank normally prevents Lmx-1 expression in this region and restricts its expression to limb-forming regions.     

Position-specific growth of mouse limb bud cells in vitro.

The relationship between cellular position and growth control has been studied in cultures of dissociated fragments of mouse limb bud cells. Using cells derived from various positions along the anterior-posterior axis of the limb bud we have developed culture conditions that optimize growth of positionally isolated cells. Under these conditions limb bud cells display an inherent, position-specific growth response; proliferation of cells derived from anterior and central regions of the limb is enhanced over that of posterior derived cells. Thus, within the total population of limb bud cells the in vitro growth of posterior cells is unique and correlates with the positional activity associated with the zone of polarizing activity. Anterior and posterior cells were cocultured to determine whether interactions between these two groups of positionally distinct cells lead to the stimulation of growth that has been observed in vivo. We observe a slight but consistent position-dependent stimulation of growth that is indicative of a mitogenic signal passing between these positionally disparate cells. Similarities between position-related growth dynamics in vivo and in vitro suggest that positional interactions that are important for limb formation can occur between dissociated cells cultured under standard conditions.     

Cell populations synthesizing cartilage proteoglycan core protein in the early chick limb bud.

Cartilage specific macromolecules are known to be synthesized in the mesenchyme of the embryonic chick limb bud, especially in areas of prechondrogenic condensations (Shinomura et al, 1984). Even though the mesenchyme seems homogeneous according to histological criteria, studies in the past have suggested the presence of different cell populations with different chondrogenic potential (Solursh et al, 1982; Swalla et al, 1984). In this study we have investigated by means of flow cytometry, the synthesis of proteoglycan core protein during early development of the chick limb bud in order to identify the different chondrocyte progenitor cells. We were able to identify by virtue of different size and density a cell population which synthesizes core protein extensively at stage 24 and stage 25 of development. This cell population synthesizes core protein predominantly at the proximal half of the limb bud at stage 24. However at stage 25 the same population synthesizes core protein predominantly at the distal half of the limb bud. These observations indicate that the distal half of stage 25 limb bud is mostly homogeneous with prechondrogenic cells and is in agreement with in vitro experiments that show high chondrogenic potential of the mesenchymal cells from this stage.     

"Fingering" the vertebrate limb

A detailed and precise picture is being pieced together about how the pattern of digits develops in vertebrate limbs. What is particularly exciting is that it will soon be possible to trace the process all the way from establishment of a signalling centre in a small bud of undifferentiated cells right through to final limb anatomy. The development of the vertebrate limb is a traditional model in which to explore mechanisms involved in pattern formation, and there is accelerating knowledge about the genes involved. One reason why the limb is holding its place in the post-genomic age is that it is rich in pre-genomic embryology. Here, we will focus on recent findings about the aspect of vertebrate limb development concerned with digit pattern across the anteroposterior axis of the limb. This process is controlled by a signalling region in the early limb bud known as the polarizing region. Interactions between polarizing region cells and other cells in the limb bud ensure that a thumb develops at one edge of the hand (anterior) and a little finger at the other (posterior).     

Temporal and spatial analysis of hyaluronidase activity during development of the embryonic chick limb bud

The glycosaminoglycan hyaluronate (HA) appears to play an important role in limb cartilage differentiation. The large amount of extracellular HA accumulated by prechondrogenic mesenchymal cells may prevent the cell-cell and/or cell-matrix interactions necessary to trigger chondrogenesis, and the removal of extracellular HA may be essential to initiate the crucial cellular condensation process that triggers cartilage differentiation. It has generally been assumed that HA turnover during chondrogenesis is controlled by the activity of the enzyme hyaluronidase (HAase). In the present study we have performed a temporal and spatial analysis of HAase activity during the progression of limb development and cartilage differentiation in vivo. We have separated embryonic chick wing buds at several stages of development into well-defined regions along the proximodistal axis in which cells are in different phases of differentiation, and we have examined HAase activity in each region. We have found that HAase activity is clearly detectable in undifferentiated wing buds at stage 18/19, which is shortly following the formation of a morphologically distinct limb bud rudiment, and remains relatively constant throughout subsequent stages of development through stage 27/28, at which time well-differentiated cartilage rudiments are present. Moreover, HAase activity in the prechondrogenic distal subridge regions of the limb at stages 22/23 and 25 is just as high as, or even slightly higher than, it is in proximal central core regions where condensation and cartilage differentiation are progressing. We have also found that limb bud HAase is active between pH 2.2 and 4.5 and is inactive above pH 5.0. This suggests that limb HAase is a lysosomal enzyme and that extracellular HA would have to be internalized to be degraded. These results indicate that the onset of chondrogenesis is not associated with the appearance or increase in activity of HAase. We suggest that possibility that HA turnover may be regulated by the binding and endocytosis of extracellular HA in preparation for its intracellular degradation by lysosomal HAase. Finally, we have found that the apical ectodermal ridge (AER)-containing distal limb bud ectoderm possesses a relatively high HAase activity. We suggest the possibility that a high HAase activity in the AER may ensure a rapid turnover and remodeling of the disorganized HA-rich basal lamina of the AER that might be essential for limb outgrowth.     

The growth and form development of the limb buds in vertebrate animals

The development of the fin and limb buds involves a balance of centrifugal (active) and centripetal (passive) mechanical forces, the first of which acts to move the walls of these structures away from each other and the second holds them together. When the volume of the mesodermal core increases, the generated force meets with the resistance of the basal membrane, and as a result, the limb bud has a tendency to acquire cylindrical shape. Collagen fibers, individual mesenchymal cells, and their groups hold together the dorsal and the ventral wall of the limb bud, prevent the movement of these walls away from each other, and in this way direct bud growth along the proximodistal and the anteroposterior axes. The balance of the forces, which stretch the ectodermal layer, and those, which constrain it, have also been observed in the development of other body parts.     

Changes in the pericellular matrix during differentiation of limb bud mesoderm

Mesodermal cells in the developing chick embryo limb bud appear morphologically homogeneous until stage 21. At stage 22 the prechondrogenic and premyogenic areas begin to condense, culminating in the appearance of cartilage and muscle by stage 25-26. We have examined changes in the hyaluronate-dependent pericellular matrices elaborated by mesodermal cells of the limb bud from different developmental stages and the corresponding changes in production of cell surface-associated and secreted glycosaminoglycans. When placed in culture, most early mesodermal cells (stage 17 lateral plate and stage 19 limb bud) exhibited pericellular coats as visualized by the exclusion of particles. These coats were removed by treatment of the cultures with Streptomyces hyaluronidase. Cells from stage 20-21 limb buds (precondensation) had smaller coats, whereas cells derived from stage 22, 24, and 26 limb buds (condensed chondrogenic and myogenic regions) lacked coats. However, coats were reformed during subsequent cytodifferentiation of chondrocytes; chondrocytes from stage 28 and 30 limb buds, and more mature chondrocytes from stage 38 tibiae, had pericellular coats. Thus, cytodifferentiation of cartilage is accompanied by extensive intercellular matrix accumulation in vivo and reacquisition of pericellular coats in vitro. Although their structure was still dependent on hyaluronate, chondrocyte coats were associated with increased proteoglycan content compared to the coats of early mesodermal cells. The amount of incorporation of [3H]acetate into cell surface hyaluronate remained relatively constant from stages 17 to 38, whereas in the medium compartment, incorporation into hyaluronate was more than 4-fold greater by stage 17 and 19 mesodermal cells than by cells from stages between 20 and 38. However, there was a progressive increase in incorporation into cell surface and medium chondroitin sulfate throughout these developmental stages. Thus, at the time of cellular condensation in the limb bud in vivo, we have observed a reduction in size of hyaluronate-dependent pericellular coats and a dramatic change in the relative proportion of hyaluronate and chondroitin sulfate produced by the mesodermal cells in vitro.     

Growth and shaping of the fin and limb buds

The development of the fin and limb buds involves a balance of centrifugal (active) and centripetal (passive) mechanical forces, the first of which acts to move the walls of these structures away from each other and the second of which holds them together. When the volume of the mesodermal core increases, the generated force meets with the resistance of the basal membrane, and as a result, the limb bud has a tendency to acquire a cylindrical shape. Collagen fibers, individual mesenchymal cells, and their groups hold together the dorsal and the ventral wall of the limb bud, prevent the movement of these walls away from each other, and in this way direct bud growth along the proximodistal and the anteroposterior axes. The balance of the forces which stretch the ectodermal layer and those which constrain it has also been observed in the development of other body parts.     

Role of FGFs in skeletal muscle and limb development

Fibroblast growth factors (FGFs) are a family of nine proteins that bind to three distinct types of cell surface molecules: (i) FGF receptor tyrosine kinases (FGFR-1 through FGFR-4); (ii) a cysteine-rich FGF receptor (CFR); and (iii) heparan sulfate proteoglycans (HSPGs). Signaling by FGFs requires participation of at least two of these receptors: the FGFRs and HSPGs form a signaling complex. The length and sulfation pattern of the heparan sulfate chain determines both the activity of the signaling complex and, in part, the ligand specificity for FGFR-1. Thus, the heparan sulfate proteoglycans are likely to play an essential role in signaling. We have recently identified a role for FGF in limb bud development in vivo. In the chick limb bud, ectopic expression of the 18 kDa form of FGF-2 or FGF-2 fused to an artificial signal peptide at its amino terminus causes skeletal duplications. These data, and the observations that FGF-2 is localized to the subjacent mesoderm and the apical ectodermal ridge in the early developing limb, suggest that FGF-2 plays an important role in limb outgrowth. We propose that FGF-2 is an apical ectodermal ridgederived factor that participates in limb outgrowth and patterning. © 1994 Wiley-Liss, Inc.     

Retinoic acid respecifies limb bud cells in vitro.

Retinoic acid (RA) is known to have dramatic effects on limb pattern formation and has been shown to exert its effects on limbs by converting anterior limb bud cells into cells with posterior positional properties. In this study we find that dissociated posterior limb bud cells from chick and mouse embryos cultured at high density (micromass cultures) are able to stimulate the formation of supernumerary digits when grafted into developing wing buds and that the positional identity of both chick and mouse limb bud cells can be maintained for finite periods of time in vitro. Furthermore, using this assay system we have tested whether anterior cells from mouse and chick limb buds can be converted into cells with posterior identity by exposure to RA in vitro. We find that anterior limb bud cells acquire posterior properties after culture in the presence of RA.     

Cell-cell interaction by mouse limb cells during in vitro chondrogenesis: Analysis of the brachypod mutation

This paper contains observations and experiments which collectively demonstrate a requirement for cell-cell interactions among limb bud mesenchyme cells during chondrogenic differentiation. Limb bud cells isolated from brachypodism^H (bp^H) and wild-type mouse embyros between Thieler stage 16–17 and midstage 21 were compared with respect to their abilities to undergo chondrogenic differentiation in high-density micromass cultures. Nodules formed by dissociated Day 12 (stage 20) bp^H limb bud cells have been reported previously to be abnormally reduced in size and number, and delayed in formation. We corroborate these results, but find that bp^H cultures prepared from earlier-stage limb buds (between stages 16–17 and early stage 21) are progressively more like wild-type cultures. Stage 16–17 bp^H cultures at 72 hr actually contain normal numbers of and size nodules, while stage 18 bp^H cultures are intermediate between stages 16–17 and stage 21 in nodule formation. On the other hand, we also find that the initial rate of aggregate formation is normal even in bp^H cultures prepared from stage 20 cultures in which nodule formation is not normal. Preparation of cultures composed primarily of early stage 21 bp^H limb bud cells mixed with small quantities (e.g., 5%) of stage 16–17 wild-type limb bud cells showed significant increases in cartilage nodule formation over control cultures composed only of early stage 21 bp^H cells. Greater proportions of wild-type cells obtained from embryos older than stages 16–17 were required for the same degree of normalization, supporting the hypothesis that a specific cell type, whose proportion decreases normally in the limb bud over time, is required to increase in vitro chondrogenesis by bp^H cells. Additionally, cultures containing stage 23 chick limb cells and early stage 21 bp^H cells at a ratio of 1:20 contained wild-type levels of nodules per square millimeter of culture. Thus, bp^H cells appear to respond to chondrogenic inductive signals from normal limb mesenchyme cells. In order to test for the ability of bp^H limb bud mesenchyme to induce chondrogenesis, stage 16–17 bp^H and wild-type limb bud cells, which form identical numbers of aggregates and nodules in culture, were each mixed with early stage 21 bp^H cells at ratios of 1:20, 1:10, and 1:3. Although low proportions of wild-type stage 17 cells significantly increased the number of aggregates and nodules in these mixed cultures, low proportions of bp^H stage 16–17 cells did not. It is, therefore, concluded that the primary defect of the bp^H mutation is likely to reside in the reduced ability of a specific mesenchyme cell subpopulation to provide an inductive stimulus for chondrogenesis.     

Control of vertebrate limb outgrowth by the proximal factor Meis2 and distal antagonism of BMPs by Gremlin

The mechanisms controlling growth and patterning along the proximal-distal axis of the vertebrate limb are yet to be understood. We show that restriction of expression of the homeobox gene Meis2 to proximal regions of the limb bud is essential for limb development, since ectopic Meis2 severely disrupts limb outgrowth. We also uncover an antagonistic relationship between the secreted factors Gremlin and BMPs required to maintain the Shh/FGF loop that regulates distal outgrowth. These proximal and distal factors have coordinated activities: Meis2 can repress distal genes, and Bmps and Hoxd genes restrict Meis2 expression to the proximal limb bud. Moreover, combinations of BMPs and AER factors are sufficient to distalize proximal limb cells. Our results unveil a novel set of proximal-distal regulatory interactions that establish and maintain outgrowth of the vertebrate limb.     

Hox-4 gene expression in mouse/chicken heterospecific grafts of signalling regions to limb buds reveals similarities in patterning mechanisms.

The products of Hox-4 genes appear to encode position in developing vertebrate limbs. In chick embryos, a number of different signalling regions when grafted to wing buds lead to duplicated digit patterns. We grafted tissue from the equivalent regions in mouse embryos to chick wing buds and assayed expression of Hox-4 genes in both the mouse cells in the grafts and in the chick cells in the responding limb bud using species specific probes. Tissue from the mouse limb polarizing region and anterior primitive streak respecify anterior chick limb bud cells to give posterior structures and lead to activation of all the genes in the complex. Mouse neural tube and genital tubercle grafts, which give much less extensive changes in pattern, do not activate 5'-located Hox-4 genes. Analysis of expression of Hox-4 genes in mouse cells in the grafted signalling regions reveals no relationship between expression of these genes and strength of their signalling activity. Endogenous signals in the chick limb bud activate Hox-4 genes in grafts of mouse anterior limb cells when placed posteriorly and in grafts of mouse anterior primitive streak tissue. The activation of the same gene network by different signalling regions points to a similarity in patterning mechanisms along the axes of the vertebrate body.     

Involvement of Wnt-5a in chondrogenic pattern formation in the chick limb bud

Members of the Wnt family are known to play diverse roles in the organogenesis of vertebrates. The full-coding sequences of chicken Wnt-5a were identified and the role it plays in limb development was examined by comparing its expression pattern with that of two other Wnt members, Wnt-4 and Wnt-11, and by misexpressing it with a retrovirus vector in the limb bud. Wnt-5a expression is detected in the limb-forming region at stage 14, and in the apical ectodermal ridge and distal mesenchyme of the limb bud. The signal was graded along the proximal-distal axis at stages 20-28 and also along the anterior-posterior axis during early stages. It disappeared in the cartilage-forming region after stage 26, and was restricted to the region surrounding the phalanges at stage 34. Wnt-4 and Wnt-11, other members of the Wnt-5a-subclass, were expressed with a distinct spatiotemporal pattern during the later phase. Wnt-4 was expressed in the articular structure and Wnt-11 was expressed in the dorsal and ventral mesenchyme adjacent to the ectoderm. Wnt-5a expression was partially reduced after apical ectodermal ridge removal, whereas Wnt-11 expression was down-regulated by dorsal ectoderm removal. Therefore, expression of these Wnt was differentially regulated by the ectodermal signal. Misexpression of Wnt-5a in the limb bud with the retrovirus resulted in truncation of long bones predominantly in the zeugopod because of retarded chondrogenic differentiation. Distal elements, such as the phalanges and metacarpals, were not significantly reduced in size. These results suggest that Wnt-5a is involved in pattern formation along the proximal-distal axis by regulation of chondrogenic differentiation.     

Hyaluronan in limb morphogenesis

Hyaluronan (HA) is a large glycosaminoglycan that is not only a structural component of extracellular matrices, but also interacts with cell surface receptors to promote cell proliferation, migration, and intracellular signaling. HA is a major component of the extracellular matrix of the distal subapical mesenchymal cells of the developing limb bud that are undergoing proliferation, directed migration, and patterning in response to the apical ectodermal ridge (AER), and has the functional potential to be involved in these processes. Here we show that the HA synthase Has2 is abundantly expressed by the distal subridge mesodermal cells of the chick limb bud and also by the AER itself. Has2 expression and HA production are downregulated in the proximal central core of the limb bud during the formation of the precartilage condensations of the skeletal elements, suggesting that downregulation of HA may be necessary for the close juxtaposition of cells and the resulting cell-cell interactions that trigger cartilage differentiation during condensation. Overexpression of Has2 in the mesoderm of the chick limb bud in vivo results in the formation of shortened and severely malformed limbs that lack one or more skeletal elements. Skeletal elements that do form in limbs overexpressing Has2 are reduced in length, exhibit abnormal morphology, and are positioned inappropriately. We also demonstrate that sustained HA production in micromass cultures of limb mesenchymal cells inhibits formation of precartilage condensations and subsequent chondrogenesis, indicating that downregulation of HA is indeed necessary for formation of the precartilage condensations that trigger cartilage differentiation. Taken together these results suggest involvement of HA in various aspects of limb morphogenesis.     

Effects of enzyme isolation on the basal lamina of developing avian limb bud epithelia

Summary The developing avian limb bud is a classic example of an epithelial-mesenchymal interaction. Numerous attempts at maintenance of the epithelia in culture have been predominantly unsuccessful. The fate of the isolated epithelial sheet of the limb bud [including the apical ectodermal ridge (AER)] in culture may depend at least in part on the integrity of its basal lamina following isolation. In this study the distal epithelium of the stage 23 limb bud was isolated utilizing trypsin and Dispase II in a variety of procedures. The integrity of the basal lamina of limb epithelium immediately upon isolation and after 2 h in culture was determined by immunofluorescent staining for laminin, and electron microscopy. In epithelial sheets isolated with Dispase II a direct relationship was observed between maintenance of the extracellular matrix at isolation and the preservation of the tissue structure and cytoarchitecture following 2 h in culture. In contrast, there was an accelerated deterioration during incubation of the tissue isolated with trypsin, independent of isolation conditions and integrity of basal lamina after isolation. Short-term maintenance of limb bud epithelial structure and cytoarchitecture after enzymatic isolation seems correlative to the maintenance of extracellular matrix at the epithelial basal surface.     

Pattern regulation in the embryonic chick limb: Supernumerary limb formation with anterior (non-ZPA) limb bud tissue

The formation of supernumerary limbs and limb structures was studied by juxtaposing normally nonadjacent embryonic chick limb bud tissue. A “wedge” (ectoderm and mesoderm) of anterior or mid donor right wing bud (stage 21) was inserted in a slit made in a host right limb bud (stage 21) at the same position as its position of origin or to a more posterior position. The AER of the donor tissue and host wing bud were aligned with each other. Donor tissue was grafted with its dorsalventral polarity the same as the host's limb bud or reversed to that of the host's. Depending on the position of origin of the donor limb bud tissue and the position to which it was transplanted in a host, supernumerary wings or wing structures formed. Furthermore, depending on the orientation of the graft in the host, supernumerary limbs with either left or right asymmetry developed. The results of experiments performed here are considered in light of two current models which have been used to describe supernumerary limb formation: one based on local, short-range, cell-cell interactions and the other based on long-range positional signaling via a diffusible morphogen.     

Association of mesenchyme with attenuated basement membranes during morphogenetic stages of newt limb regeneration

The interface between epithelium and mesenchyme may be involved in inductive interactions which occur during development. This interface within the growth bud, or blastema, of a regenerating limb has been examined to determine whether changes in basement-membrane structures are visible in regions of putative epithelial-mesenchymal inductive interaction. Regenerating forelimbs of adult newts were fixed by perfusion with osmotically balanced aldehydes. Late-bulb to early-digit stage regenerates were collected and processed either for light and transmission electron microscopy or for scanning electron microscopy. Light microscopy confirmed that regions characterized by increased numbers of subepithelial mesenchymal cells were covered by a diffusely stained basement membrane. Transmission electron microscopy of these regions revealed two structural components of the basement membrane. The thin basal lamina was continuous in all regions of all stages examined, but it was attenuated apically in areas of mesenchymal cell accumulation. The thicker underlying reticular lamina was markedly attenuated in these regions near the blastemal apex. Scanning electron microscopy of de-epithelialized blastemas revealed that, apically, the reticular lamina formed only a delicate lacelike network. On the base of the blastema, it formed a dense fibrillar meshwork which was further organized into a geometric pattern in the adjacent stump skin. Cumulatively, these observations suggest that physical contact between epithelial and mesenchymal cells is not essential at these stages, but that regions of putative epithelial-mesenchymal interaction are characterized by a distinctly diminished reticular lamina. Structural changes in basement-membrane components may be related to termination of local inductive events.     

The recombinant limb as a model for the study of limb patterning, and its application to muscle development

The recombinant limb is a model system that has proved fruitful for analyzing epithelial-mesenchymal interactions and understanding the functional properties of the components of the limb bud. Here we present an overview of some of the insights obtained through the use of this technique. Among these are the understanding that fore or hind limb identity is inherent to the limb bud mesoderm, that the apical ectodermal ridge (AER) is a permissive signaling center and that the limb bud ectoderm plays a central role in the control of dorsoventral polarity. Recombinant limb studies have also allowed the identification of the affected tissue component in several limb mutants. More recently this model has been applied to the study of regulation of gene expressions related to patterning. In this report we use recombinant limbs to analyze pattering of the Pax3 expressing limb muscle cell lineage in the early stages of limb development. In recombinant limbs made without the zone of polarizing activity (ZPA), myoblasts appear intermingled with other mesodermal cells at the beginning of the recombinant limb development. Rapidly thereafter, the muscle precursors segregate and organize around the central forming chondrogenic core of the recombinant. Although this segregation is reminiscent of that occurring during normal development, the myoblasts in the recombinant fail to proliferate appropriately and also fail to migrate distally. Consequently, the muscle pattern in the recombinant limb is defective indicating that normal patterning cues are absent. However, recombinant limbs polarized with a ZPA exhibited a larger mass of muscle cells and a more normal morphogenesis, supporting a role for this signaling center in limb muscle development. Finally, we have ruled out host somite contributions to recombinant limbs by grafting chick recombinant limbs to quail hosts. This initial report demonstrates the value of the recombinant limb model system for dissecting the environmental cues required for normal muscle limb patterning. Received: 31 August 1998 / Accepted: 29 September 1998