Gap junctional communication during limb cartilage differentiation

The onset of cartilage differentiation in the developing limb bud is characterized by a transient cellular condensation process in which prechondrogenic mesenchymal cells become closely apposed to one another prior to initiating cartilage matrix deposition. During this condensation process intimate cell-cell interactions occur which are necessary to trigger chondrogenic differentiation. In the present study, we demonstrate that extensive cell-cell communication via gap junctions as assayed by the intercellular transfer of lucifer yellow dye occurs during condensation and the onset of overt chondrogenesis in high density micromass cultures prepared from the homogeneous population of chondrogenic precursor cells comprising the distal subridge region of stage 25 embryonic chick wing buds. Furthermore, in heterogeneous micromass cultures prepared from the mesodermal cells of whole stage 23/24 limb buds, extensive gap junctional communication is limited to differentiating cartilage cells, while the nonchondrogenic cells of the cultures that are differentiating into the connective tissue lineage exhibit little or no intercellular communication via gap junctions. These results provide a strong incentive for considering and further investigating the possible involvement of cell-cell communication via gap junctions in the regulation of limb cartilage differentiation.     

The role of gap junctions in patterning of the chick limb bud

The role of gap junctional communication during patterning of the chick limb has been investigated. Affinity-purified antibodies raised against rat liver gap junctional proteins were used to block communication between limb mesenchyme cells. Co-injection of the antibodies and Lucifer yellow into mesenchyme cultures demonstrated that communication was inhibited almost immediately. When antibodies were loaded into mesenchyme tissue by DMSO permeabilization, [3H]nucleotide transfer was prevented for at least 16 h. Polarizing region tissue from the posterior limb bud margin causes digit duplications when grafted to the anterior margin. Quail polarizing region cells were loaded with gap junction antibody and grafted into chick wing buds. The antibody had no effect on growth or survival of the grafted cells. As very few polarizing region cells are required to initiate duplications, the number of polarizing region cells in the grafts was reduced by diluting 1:9 with anterior mesenchyme tissue. When either polarizing region or anterior mesenchyme tissue in the graft was loaded separately with antibody, there was little effect on respecification of the digit pattern. However, loading both tissues in the graft caused a significant decrease in duplications. This indicates that a major role of gap junctions in limb patterning may be to enable polarizing region cells to communicate directly with adjacent anterior mesenchyme. A role for gap junctional communication between anterior mesenchyme cells cannot be excluded. The results are discussed in relation to the role of retinoic acid as a putative morphogen.     

Extravascular fluid dynamics of the embryonic chick wing bud

While a number of models of positional information in the chick wing bud have involved the diffusion of morphogens to establish chemical gradients of morphogenetic activity, only recently have there been attempts to characterize the milieu in which such diffusion must take place. We report an analysis of the fluid dynamics of the extravascular (interstitial) spaces of stage 22-25 chick wing buds, into which aqueous aniline blue dye was microinjected as a visible, unreactive tracer. Six sites along the antero-posterior (A-P) and proximo-distal (P-D) axes were chosen for study. Injections of dye into the posterior half of the wing bud exhibited marked directionality of extravascular transport (mean of all posterior sites = 68%), while anterior injections showed little or no directionality (mean of all anterior sites = 13%). All instances of directed dye movement were disto-proximal, the same direction as the blood flow through the marginal veins. In embryos gassed in situ with CO2, which reversibly stopped the heartbeat and vascular flow, directionality was abolished, yet diffusion rates were unaffected. Posterior disto-proximal extravascular dye movement was correlated with the greater diameter, flow velocity, and volume flow rate of the posterior marginal vein, compared to the anterior marginal vein. Radial diffusion rates were measured, and posterior disto-proximal rates were corrected for measured disto-proximal directionality by the use of a simple diffusion-translation model. Three-way analysis of variance showed that directionality-uncorrected disto-proximal rates in posterior sites were not significantly different from anterior radial rates, but that directionality-corrected posterior rates did differ significantly (P less than 0.0001). A significant stage effect (P less than 0.005) and a significant interaction between the A-P axis and stage (P less than 0.05) were also found. We hypothesize that the spatial arrangement and flow patterns of the vasculature physically determine the fluid dynamics of the interstitium. Based on these observations, we also suggest that disto-proximal extravascular fluid movement in the posterior wing bud presents a barrier to the free diffusion of aqueous molecules, including morphogens originating in the "zone of polarizing activity."     

Posterior apical ectodermal ridge removal in the chick wing bud triggers a series of events resulting in defective anterior pattern formation

The ability of the anterior apical ectodermal ridge to promote outgrowth in the chick wing bud when disconnected from posterior apical ridge was examined by rotating the posterior portion of the stage-19/20 to stage-21 wing bud around its anteroposterior axis. This permitted contact between the anterior and posterior mesoderm, without removing wing bud tissue. In a small but significant number of cases (10/54), anterior structures (digit 2) formed spatially isolated from posterior structures (digits 3 and 4). Thus, continuity with posterior ridge is not a prerequisite for anterior-ridge function in the wing bud. Nevertheless, posterior-ridge removal does result in anterior limb truncation. To investigate events leading to anterior truncation, we examined cell death patterns in the wing bud following posterior-ridge removal. We observed an abnormal area of necrosis along the posterior border of the wing bud at 6-12 h following posterior-ridge removal. This was followed by necrosis in the distal, anterior mesoderm at 48 h postoperatively and subsequent anterior truncation. Clearly, healthy posterior limb bud mesoderm is needed for anterior limb bud survival and development. We propose that anterior truncation is the direct result of anterior mesodermal cell death and that this may not be related to positional specification of anterior cells. In our view, cell death of anterior mesoderm, after posterior mesoderm removal, should not be used as evidence for a role in position specification by the polarizing zone during the limb bud stages of development. We suggest that the posterior mesoderm that maintains the anterior mesoderm need not be restricted to the mapped polarizing zone, but is more extensively distributed in the limb bud.     

Temporal pattern of posterior positional identity in mouse limb buds

This study describes the temporal pattern of posterior positional identity in mouse limb bud cells. To do this wedges of tissue from the posterior edge of mouse limb buds at various stages (limb stages: Wanek et al., 1989b. J. Exp. Zool. 249, 41-49) were grafted to the anterior edge of a host chick embryo wing bud. Grafts of mouse posterior cells are able to induce the formation of supernumerary digits every time when they are taken from buds from stage 3 through stage 6. At stage 7, the frequency declines and by stage 8 the chick cells no longer respond. The results indicate a change in tissue properties at stage 7, which progresses by stage 8 to the point at which posterior positional identity is no longer detectable by this assay. These temporal changes in this aspect of limb pattern formation can be used as an additional criterion to guide the identification of genes involved in the specification of posterior positional identity.     

The in vitro maintenance of the apical ectodermal ridge of the chick embryo wing bud: an assay for polarizing activity.

We have devised an in vitro bioassay for limb bud polarizing activity in the chick embryo. This assay has proven to be a relatively quick and effective test for a morphogenetic factor asymmetrically distributed in the limb bud which is capable of maintaining or thickening the apical ectodermal ridge.A small section of the preaxial border of the chick embryo wing bud was cultured alone, with tissue from the posterior border, mid-dorsal or anterior corner of a second donor wing, or from the flank. The tissue from the preaxial border (responding tissue) consisted of mesoderm with overlying ectoderm and apical ectodermal ridge. When the responding tissue was cultured alone, with flank, or with anterior corner limb tissue, the apical ectodermal ridge flattened in 24–36 hr and many macrophages appeared in the underlying mesoderm. When cultured with posterior border limb tissue however, the apical ridge of the responding tissue remained thickened for up to 48 hr., and no macrophages appear in the underlying mesoderm. The behavior of responding tissue was intermediate between these two extremes when cultured with mid-dorsal limb tissue. The morphogenetic activity assayed by this procedure thus seems to be present as a gradient in the wing bud, with activity decreasing from posterior to anterior. Contact with the responding tissue is not required to enable posterior border tissue to elicit ridge thickening and inhibit the cell death.     

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.     

Distribution of Retinoids Applied Exogenously to Chick Limb Buds: an Autoradiographic Analysis

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.     

The ability of the chick wing bud to regulate positional disparity along the anterior-posterior axis

When wedges of wing bud tissue are added to a host wing bud so there is positional disparity between graft and host, skeletal duplications result (L. E. Iten and D. J. Murphy 1980) Dev Biol. 75, 373-385. The polarity of the duplications is predictable by the polar coordinate model, leading to the interpretation that the positional disparity caused the duplications. To determine whether positional disparity alone causes duplications, without the complication of added tissue, we rotated wedges of ectoderm and mesoderm around the proximodistal axis within the wing bud. Wedges measuring 200-800 micron along the distal edge were rotated 180 degrees at stages 20-22, reversing the anteroposterior and dorsoventral axes relative to the bud. This caused positional disparity, similar to that achieved by Iten and Murphy (1980), without the addition of tissue. We found that rotations involving no polarizing zone tissue produced normal wings or wings lacking some distal parts, as did rotations of tissue lying entirely within the polarizing zone. However, when polarizing zone mesoderm was displaced, so that polarizing and nonpolarizing tissues were juxtaposed, a majority of the operations produced polarized skeletal duplications. Our data demonstrate that positional disparity alone does not cause skeletal duplications in the chick wing bud, unless polarizing zone tissue is displaced. Further, these data demonstrate that the chick wing bud can regulate to form a normal wing skeleton in the face of large positional disparity, provided that the polarizing zone is not moved. Finally, our results may be explained by the action of the proposed polarizing morphogen on the displaced cells causing repolarization.     

Position of origin of donor posterior chick wing bud tissue transplanted to an anterior host site determines the extra structures formed

The formation of supernumerary limb structures was studied by juxtaposing normally nonadjacent embryonic chick limb bud tissue. Different “wedges” (ectodern and mesoderm) of posterior donor right wing bud (stage 21) were transplanted to a slit made in stage 20–23 host right wing buds. Donor posterior tissue was transplanted to an anterior position in a host wing bud or, as a control, to the same position as its position of origin. Transplanting different wedges of posterior tissue to the same anterior host position results in wings with supernumerary structures, and different extra structures form depending on the position of origin of the donor tissue. The identification of extra limb structures formed was based on the skeletal and integumentary patterns of resulting wings and the pattern of muscles as seen in serial sections of resulting limbs. The results of experiments presented here are considered in light of current models that have been used to describe the formation of supernumerary limb structures by the embryonic chick limb bud.     

Polarizing activity in the developing limb of the Syrian hamster.

The transplantation of small pieces of tissue from the limb buds of 9 1/2 -10 day hamster embryos to the wing bud of the chick results in the induction of supernumerary wing structures. The anteroposterior polarity of these induced structures is under the control of the transplanted hamster tissue. The developing hamster limb thus has limb polarizing activity similar to that found in avian species and, as in the chick, the activity is found primarily in the posterior region of the limb bud.     

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.     

Cell adhesiveness and affinity for limb pattern formation

Stage-dependent cell sorting in vitro is an intriguing property that mesenchymal cells of a chick limb bud have. We previously proposed that N-cadherin, a cell adhesion molecule, is involved in the sorting process and is likely to be a component of the mechanism of proximal-distal patterning in the developing limb (Yajima et al., (1999) Dev. Dynam. 216:274-284). Here, we present more direct evidence that N-cadherin is one of the molecules responsible for regulation of stage-dependent cell sorting in vitro. Our results suggest that N-cadherin, which accumulates in the distal region of the chick limb bud as limb development proceeds, is related to the positional identity that gives rise to the different shapes and numbers of cartilaginous elements along the proximal-distal axis. In this article we also give insights into positional identity which is mediated by Hoxgenes and cell surface property during limb development.     

Ischemic Preconditioning Protects Against Gap Junctional Uncoupling in Cardiac Myofibroblasts

Ischemic preconditioning increases the heart's tolerance to a subsequent longer ischemic period. The purpose of this study was to investigate the role of gap junction communication in simulated preconditioning in cultured neonatal rat cardiac myofibroblasts. Gap junctional intercellular communication was assessed by Lucifer yellow dye transfer. Preconditioning preserved intercellular coupling after prolonged ischemia. An initial reduction in coupling in response to the preconditioning stimulus was also observed. This may protect neighboring cells from damaging substances produced during subsequent regional ischemia in vivo, and may preserve gap junctional communication required for enhanced functional recovery during subsequent reperfusion.     

Ischemic preconditioning protects against gap junctional uncoupling in cardiac myofibroblasts

Ischemic preconditioning increases the heart's tolerance to a subsequent longer ischemic period. The purpose of this study was to investigate the role of gap junction communication in simulated preconditioning in cultured neonatal rat cardiac myofibroblasts. Gap junctional intercellular communication was assessed by Lucifer yellow dye transfer. Preconditioning preserved intercellular coupling after prolonged ischemia. An initial reduction in coupling in response to the preconditioning stimulus was also observed. This may protect neighboring cells from damaging substances produced during subsequent regional ischemia in vivo, and may preserve gap junctional communication required for enhanced functional recovery during subsequent reperfusion.     

Retinaldehyde, a potent inhibitor of gap junctional intercellular communication

Retinaldehyde and retinoic acid are derivatives of vitamin A, and retinaldehyde is the precursor for the synthesis of retinoic acid, a well-known inhibitor of gap junctional intercellular communication. In this investigation, we asked the question if retinaldehyde has similar effects on gap junctions. Gap junctional intercellular communication was measured by scrape-loading and preloading dye-transfer methods, and studies were carried out mainly on cultured liver epithelial cells. Retinaldehyde was found to be a more potent inhibitor (dye transfer reduced by 50% at 2.8 μM) than retinoic acid (dye transfer reduced by 50% at 30 μM) and glycyrrhetinic acid (dye transfer reduced by 50% at 65 μM). Both the 11-cis and all-trans forms of retinaldehyde were equally effective. Retinaldehyde inhibited dye transfer of both anionic Lucifer yellow and cationic Neurobiotin. Inhibition by retinaldehyde developed in less than two minutes at 50 μM, but unlike the reported case with retinoic acid, recovery was slower, though full. In addition to liver epithelial cells, retinaldehyde inhibited gap junctional communication in lens epithelial cells, retinal pigment epithelial cells and retinal ganglion cells.     

Formation of supernumerary structures by the embryonic chick wing depends on the position and orientation of a graft in a host limb bud

The relationship between the position transplanted in a host limb bud, the orientation of a graft in a host limb bud, and the extra limb structures formed was studied by juxtaposing normally nonadjacent embryonic chick wing bud tissue. In one series of transplantation operations, two different wedges (ectoderm and mesoderm) of stage 21 right donor posterior wing bud tissue were transplanted to the middle of a host stage 20 to 22 right wing bud such that the dorsal-ventral polarity of the graft and host were the same or reversed. The results of these transplantation operations show that the formation of supernumerary limb structures depends on the position of origin of the donor tissue, the anterior-posterior position transplanted in a host limb bud, and the orientation of the graft in the host limb bud. In a second series of transplantation operations, the relationship between the proximodistal position where posterior donor tissue is transplanted in an anterior host site and the extra structures formed was studied. A wedge of posterior stage 21 right wing bud tissue was transplanted to an anterior proximal or anterior distal site of a stage 22 to 24 host right wing bud. The results of these transplantation operations show that when the donor tissue is transplanted to an anterior proximal position in a host wing bud, then limbs with only a duplicated humerus result, whereas, when transplanted to an anterior distal position, then limbs with a duplicated forearm element and extra digits result.