DEVELOPMENTAL
BIOLOGY
148, 529-535
(1991)
along the AnteriorA Gradient of Gap Junctional Communication Posterior Axis of the Developing Chick Limb Bud CAROLINEN. Department
qf Anatomy,
Uni?lersity
D. COELHOANDROBERTAKOSHER of Connecticut Accepted
Health
August
Center,
Farmingfcm,
Connecticut
06030
2.9, 19.91
A modification of the scrape-loading/dye transfer technique was used to study gap junctional communication along the anterior-posterior (A-P) axis of embryonic chick wing buds at an early stage of development (stage 20121) when positional values along the A-P axis are being specified. Extensive intercellular transfer of the gap junction-permeable dye, lucifer yellow, from scrape-loaded mesenchymal cells to contiguous cells occurs in the posterior mesenchymal tissue of the wing bud adjacent to the zone of polarizing activity, which is thought to be the source of a diffusible morphogen that specifies A-P positional identity according to its local concentration. Considerably less transfer of lucifer yellow dye occurs in scrape-loaded mesenchymal tissue in the middle of the limb bud compared to posterior mesenchymal tissue, and little or no transfer of lucifer yellow is observed in the mesenchymal tissue in the anterior portion of the limb bud. No intercellular transfer of the gap junction-impermeable dye, rhodamine dextran, occurs in any region of the limb bud. These results indicate that there is a gradient of gap junctional communication along the A-P axis of the developing chick wing bud. This gradient of gap junctional communication along the A-P axis might generate a graded distribution of a relatively low molecular weight intracellular regulatory molecule involved in specifying A-P positional identities. ,2’ 1991 Academic Press, Inr. INTRODUCTION
A problem of major importance in development concerns the cellular and molecular mechanisms involved in generating the three-dimensional spatial organization of the various morphologically distinct skeletal elements of the limb in their appropriate position and sequence along the anterior-posterior (A-P) axis. A small group of mesenchymal cells located at the posterior margin of the developing limb bud appear to play an important role in regulating pattern formation along the A-P axis. When cells from this posterior region, which is known as the zone of polarizing activity (ZPA), are transplanted to the anterior margin of a host limb bud, they elicit the formation of duplicated distal skeletal elements from the host tissue which exhibit mirror image symmetry to the normal elements (Saunders and Gasseling, 1968). Several observations suggest that the ZPA is the source of a diffusible morphogen which forms a concentration gradient across the A-P axis of the limb, and that high concentrations of the morphogen specify the formation of posterior structures, while progressively lower concentrations specify the formation of progressively more anterior structures (Tickle et al., 1975). Several recent studies have provided evidence indicating that retinoic acid may be the putative morphogen released by the ZPA (see Brickell and Tickle, 1989; Eichele, 1989, for reviews). Implantation of beads soaked in retinoic acid into the anterior margin of the limb bud provokes the same mirror-image duplication 529
of skeletal elements as does implantation of ZPA tissue (Tickle et ah, 1982,1985). Moreover, endogenous retinoic acid is distributed in a graded fashion across the A-P axis of the limb bud such that its concentration is 2.5fold higher in the ZPA-containing posterior quarter of the limb bud than in the anterior portion (Thaller and Eichele, 1987). Furthermore, nuclear receptors and cytoplasmic binding proteins for retinoic acid are present in the developing limb bud (Maden et aZ., 1988; Dolle et al., 1989b). It should be noted, however, that recent studies suggest that retinoic acid may function by establishing or inducing the formation of the ZPA, which in turn might produce another factor that more directly specifies A-P positional identities (Noji et al., 1991; Wanek et al., 1991). Ultrastructural studies have revealed the presence of gap junctions between limb mesenchymal cells at early stages of limb development (Kelley and Fallon, 1978, 1983). Furthermore, grafts composed of ZPA tissue and anterior limb mesenchymal cells that had been loaded with antibodies against rat liver gap junctional proteins are considerably impaired in their ability to elicit the formation of duplicated skeletal elements when implanted into the anterior margin of limb buds, suggesting the possibility that gap junctional communication may be involved in regulating pattern formation (Allen et cd., 1990). In the present investigation, we have studied gap junctional communication along the A-P axis of embryonic chick wing buds at an early stage of development in OOlZ-1606/91 Copyright All rights
$3.00
W 1991 by Academic Press, Inc. uf reproduction in any form reserved.
530
DEVELOPMENTALBIOLOGY V0~~~~148,1991
which positional values along the A-P axis are being specified. We have found that extensive gap junctional communication occurs among posterior mesenchymal cells of the limb bud that are adjacent to the ZPA, and that progressively less intercellular communication occurs among mesenchymal cells in progressively more anterior regions of the limb bud. This gradient of gap junctional communication along the A-P axis suggests that intercellular signalling via gap junctions may be involved in specification of pattern along the A-P axis of the developing limb. MATERIALS
AND
METHODS
Gap junctional communication was assayed by monitoring the intercellular transfer of lucifer yellow dye using a modification of the scrape-loading/dye transfer technique (El-Fouly et al, 1987). Wing buds were removed from stage 20/21 (Hamburger and Hamilton, 1951) White Leghorn chick embryos, and the limb mesenchyme at various locations along the A-P axis was incised perpendicular to the apical ectodermal ridge or to the base of the limb with a fine scalpel blade. The incised limb buds were then incubated in a 0.05% solution of the gap junction-permeable dye, lucifer yellow (Sigma) and the gap junction-impermeable dye, rhodamine dextran (Molecular Probes, Inc.) in phosphatebuffered saline (PBS). Transfer of lucifer yellow from the incised cells to contiguous cells via gap junctions was allowed to proceed for 2-20 min, after which the tissues were washed with PBS; fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4; washed with PBS; and examined by fluorescence microscopy either before or after being mounted under cover slips in 10% glycerin in PBS. In order to effectively and consistently observe differential gap junctional communication along the A-P axis, it is important that the incisions through the mesenthyme in different regions of the same limb bud be as uniform in depth as possible. In particular, the depth of the incisions should extend about one-half of the way through the mesenchyme, since inconsistent results are obtained when the incisions are too superficial, and excessive damage and distortion of the tissue occurs when the mesenchyme is completely sliced. The uniformity of
the incisions in the different regions of the limb mesenthyme can be judged by comparing the extent of loading of the gap junction-impermeable dye, rhodamine dextran, into the wounded cells at the edge of the incisions. Only specimens in which comparable loading of rhodamine dextran was observed were used to evaluate the extent of lucifer yellow dye transfer in the different regions of the limb mesenchyme. Of the hundreds of samples we have attempted, we estimate that we can obtain uniform incisions in the different limb regions at least 60% of the time, and when such incisions are achieved, the gradient of dye transfer is clearly evident at least 90% of the time. RESULTS
Gap junctional communication as assayed by the intercellular transfer of lucifer yellow dye was monitored at various locations along the A-P axis of stage 20/21 wing buds, a stage of development in which positional values along the A-P axis are being specified. As shown in Fig. 1, during a 2-min incubation period extensive intercellular transfer of lucifer yellow dye from scrapeloaded mesenchymal cells to contiguous cells occurs in the posterior mesenchymal tissue adjacent to the ZPA, indicating extensive gap junctional communication is occurring in the posterior portion of the limb bud. Considerably less transfer of lucifer yellow dye occurs in scrape-loaded mesenchymal tissue in the middle of the limb bud compared to posterior mesenchymal tissue (Fig. l), and little transfer of lucifer yellow is observed in the mesenchymal tissue in the anterior portion of the limb bud (Fig. 1). The distance traversed by the dye during the 2-min incubation period ranges from about 1.5to 2.5-fold greater in the posterior mesenchymal tissue than in the mesenchymal tissue in the middle of the limb and from about 3- to 12-fold greater than in the anterior mesenchymal tissue. A similar gradient of dye transfer along the A-P axis was observed when scrapeloaded limb buds were incubated in lucifer yellow for up to 20 min. As illustrated in Fig. 2, lucifer yellow dye is located intracellularly throughout the cells contiguous to the incision in posterior mesenchyme. This observation indicates that lucifer yellow is being transferred from cell to
FIG. 1. Fluorescence (A-F) and corresponding bright light (a-f) photomicrographs illustrating the extent of intercellular transfer of lucifer yellow dye via gap junctions among mesenchymal cells in various regions along the anterior-posterior axis of stage 20/21 embryonic chick wing buds. The posterior border of the limb buds is indicated by the letter P and the anterior border is indicated by the letter A. Note that extensive intercellular transfer of lucifer yellow from scrape-loaded mesenchymal cells to contiguous cells occurs in the posterior mesenchymal tissue that is adjacent to the zone of polarizing activity (ZPA) which is located at the posterior margin of the limb buds. Considerably less transfer of lucifer yellow occurs in scrape-loaded mesenchymal tissue in the middle of the limb buds, and little transfer of the dye occurs in the mesenchyma1 tissue in the anterior portion of the limb bud. x50.
532
DEVELOPMENTALBIOLOGY
V0~~~~148,1991
COELHOANDKOSHER
Grip Ju~fiot~s
cell, and that the differential pattern of dye transfer along the A-P axis reflects differences in gap junctional communication in the various limb regions. Furthermore, as shown in Figs. 3A and 3B, the gap junction-impermeable dye, rhodamine dextran, remains localized in cells at sites of incision and is not transferred to contiguous cells. As shown in Figs. 3C and 3D, when scrapeloaded limb buds were fixed within seconds after being exposed to lucifer yellow dye, comparable amounts of the dye were localized at the sites of incision in all regions of the limb bud, indicating that comparable amounts of the dye are initially loaded into the incised cells in the different regions of the limb mesenchyme. This observation indicates that there are not major differences in the extent of dye loading in the different limb regions and that loading occurs to a sufficient extent in a reasonably comparable number of cells to evaluate reliably the dramatic differences in the extent of lucifer yellow transfer to contiguous cells in the different regions of the limb bud. DISCUSSION
The results of the present study indicate that there is a gradient of gap junctional communication along the A-P axis of the developing chick wing bud, with extensive intercellular communication occurring among posterior mesenchymal cells adjacent to the ZPA and progressively less communication occurring among mesenchymal cells in progressively more anterior regions of the limb bud. The observed gradient of gap junctional communication along the A-P axis and its relationship to the ZPA suggests that intercellular signalling via gap junctions may be involved in specification of positional values along the A-P axis of the developing limb. A gradient of gap junctional communication along the A-P axis would be an excellent mechanism for generating a graded distribution of a relatively low molecular weight intracellular regulatory molecule involved in specifying A-P positional values. It can be argued that gap junctions that might be present among anterior mesenchymal cells might be shut down in response to damage elicited by scrape-loading, whereas the gap junctions among the posterior mesenchymal cells might not be shut down in response to the scrape-loading procedure. Although this possibility is difficult to conclusively eliminate, the fact that little or no lucifer yellow dye transfer is detectable among the
FIG. 2. High magnification fluorescence photomicrograph bud. Lucifer yellow dye is located intracellularly throughout from cell to cell.
trrd
Limb
Paffrrn
Formcxfiotc
533
anterior mesenchymal cells regardless of if the cells are incubated in the dye for a few seconds or up to 20 min argues against it. The fact that little or no transfer of lucifer yellow is detectable in the anterior mesenchyme when the cells are fixed within seconds after being exposed to the dye argues against the possibility that extremely rapid transfer and dissipation of the dye is occurring among the anterior mesenchymal cells. The gradient of gap junctional communication along the A-P axis of the limb bud that we have observed combined with the studies of Allen et al. (1990) provides strong incentive for considering and further investigating the role of gap junctional signalling in the regulation of pattern formation along the A-P axis. Our observations are consistent with the suggestions that close range cell-cell interactions are involved in regulating positional identities (French et al., 1976; Bryant et al., 1986; Wanek et al., 1991). Considerable evidence has accumulated suggesting that retinoic acid may be the diffusible morphogen released by the ZPA that becomes distributed in a graded fashion across the A-P axis of the limb bud and specifies positional values along the axis (Brickell and Tickle, 1989; Eichele et aZ., 1989). It is therefore of considerable interest that retinoic acid is a potent modulator of gap junctional communication in several cell types (Hossain et al, 1989; Mehta et al., 1989). In particular, higher concentrations of retinoic acid enhance gap junctional communication, while lower concentrations of retinoic acid inhibit intercellular communication (Hossain et al., 1989; Mehta et al., 1989). It is therefore conceivable that the higher concentrations of retinoic acid present in the posterior region of the limb bud may promote the extensive gap junctional communication that is occurring in this region, while the lower concentrations of retinoic acid in the anterior region of the limb bud may be involved in inhibiting cell-cell communication. Retinoic acid may thus generate a gradient of gap junctional communication across the A-P axis that in turn establishes a gradient of a gap junction-permeable regulatory molecule that specifies positional values. This suggestion interfaces well with the recent studies indicating that retinoic acid might act by establishing or inducing the formation of the ZPA, which, in turn, might produce another factor that more directly specifies A-P positional identities (Noji el ah, 1991; Wanek et al., 1991). In addition to the gradients of gap junctional communication and retinoic acid, certain homeobox-containing
of scrape-loaded mesenchymal cells in the posterior region of a stage 20/21 wing the cells contiguous to the incision, indicating that the dye is being transferred
DEVELOPMENTAL BIOLOGY
VOLUME 148.1991
tion and/or retinoic acid concentrations might regulate the differential pattern of expression of homeobox-containing genes, or vice versa, must be considered. All of these observations are consistent with the possibility that interacting opposing gradients of expression of various molecules may be involved in determining positional values along the A-P axis during limb development. This research was supported by NIH Grants HD22896 and HD22610 to R.A.K. REFERENCES
FIG. 3. (A and B) The posterior mesenchyme of a scrape-loaded stage 20/21 wing bud that had been incubated for 2 min in the presence of the gap junction-impermeable dye, rhodamine dextran (A) and the gap junction-permeable dye, lucifer yellow (B). Rhodamine dextran-labeled cells are limited to the site of incision (A), whereas lucifer yellow is transferred to contiguous cells (B). (C) A scrape-loaded stage 20/21 wing bud that was fixed within seconds after being exposed to lucifer yellow dye. Note that comparable amounts of the dye are localized at the sites of incision in all regions of the limb bud, indicating that comparable amounts of the dye are initially loaded into the incised cells in the different regions of the limb bud mesenthyme. (D) A higher magnification photomicrograph illustrating that lucifer yellow is indeed loaded into incised cells in the anterior mesenthyme.
genes, which have been implicated in the regulation of positional identity during development, are also expressed in a graded fashion along the A-P axis of the developing limb bud. For example, the chicken homeobox-containing gene GHox-8 (Coelho et al, 1991) and the chicken cognate of the Xenopus XlHbox 1 gene (the homolog of mouse Hox-3.3) (Oliver et al., 1988) are expressed predominantly in the anterior compartment of the distal mesoderm of chick limb buds, whereas several members of the HOX-4 cluster of homeobox-containing genes exhibit the complementary pattern of expression, being predominantly expressed in the ZPA-containing posterior mesoderm of the limb bud (Dolle et ah, 1989a; Oliver et al., 1989; Nohno et al, 1991). Therefore, the possibility that differential gap junctional communica-
ALLEN, F., TICKLE, C., and WARNER, A. (1990). The role of gap junctions in patterning of the chick limb bud. Development 108,623-634. BRICKELL, P. M., and TICKLE, C. (1989). Morphogens in chick limb development. BioEssoys 11, 145-149. BRYANT, S. V., and MUNEOKA, K. (1986). Views of limb development and regeneration. Trends Geuef. 2, 153-159. COELHO, C. N. D., SUMOY, L., RODGERS, B. J., DAVIDSON, D. R., HILL, R. E., UPHOLT, W. B., and KOSHER, R. A. (1991). Expression of the chicken homeobox-containing gene GHox-8 during embryonic chick limb development. Mmh. Dev. 34, 143-154. DOLLE, P., IZPISUA-BELMONTE, J.-C., FALKENSTEIN, H., RENUCCI, A., and DUBOULE, D. (1989a). Coordinate expression of the murine Hox5 complex homeobox-containing genes during limb pattern formation N~~turf342, 767-772. DOLLE, P., RUBERTE, E., KASTNER, P., PETKOVICH, M., STONER, C. M., GUDAS, L. J., and CHAMBON, P. (198913). Differential expression of genes encoding (Y,6, and gamma retinoic acid receptors and CRABP in the developing limbs of the mouse. Nuture 342, 702-705. EICHELE, G. (1989). Retinoids and vertebrate limb pattern formation. Trexds Gem?. 5, 246-251. EL-FOULY, M. H., TROSKO, J. E., and CHANG, C.-C. (1987). Scrape-loading and dye transfer. A rapid and simple technique to study gap junctional intercellular communication. Exp. Cell Res. 168,422-430. FRENCH, V., BRYANT, P. J., and BRYANT, S. V. (1976). Pattern regulation in epimorphic fields. Sciv~~cr 193,969-981. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Mo&ol. 88,49-92. HOSSAIN, M. Z., WILKINS, L. R., MEHTA, P. P., LOEWENSTEIN, W., and BERTRAM, J. S. (1989). Enhancement of gap junctional communication by retinoids correlates with their ability to inhibit neoplastic 10, 1743-1748. transformation. Ctrrci~togenesis KELLEY, R. 0.. and FALLON, J. F. (1978). Identification and distribution of gap junctions in the mesoderm of the developing chick limb bud. J Ew/hr~ol. Es!). Mw$vhd. 46, 99-110. KELLEY, R. O., and FALLON, J. F. (1983). A freeze-fracture and morphometric analysis of gap junctions of limb bud cells: Initial studies on a possible mechanism for morphogenetic signalling during development. 17~“Limb Development and Regeneration, Part A” (J. F. Fallon and A. I. Caplan, Eds.), pp. 119-130. Liss, New York. MADEN, M., ONG, D. E., SUMMERBELL, D., and CHYTIL, F. (1988). Spatial distribution of cellular protein binding to retinoic acid in the chick limb bud. Nature 335, 733-735. MEHTA, P. P., BERTRAM, J. S., and LOEWENSTEIN, W. R. (1989). The action of retinoids on cellular growth correlate with their actions on gap junctional communication. J. Cell Biol. 108, 1053-1065. NOHNO, T., NOJI, S., KOYAMA, E., OHYAMA, K., MYOKAI, F., KUROIWA, A., SAITO, T., and TANIGUCHI, S. (1991). Involvement of the Chox-4 chicken homeobox genes in determination of anteroposterior axial polarity during limb development. Cell 64, 1197-1205.
COELHO
AND KOSHER
Gup Junctirms and Limb Pattern Formaticun
NOJI, S., NOHNO, T., KOYAMA, E., MUTO, K., OHYAMA, K., AOKI, Y., TAMURA, K., OHSUGI, K., IDE, H., TANIGUCHI, S., and SAITO, T. (1991). Retinoic acid induces polarizing activity but is unlikely to be a morphogen in the chick limb bud. Nature 350,83-86. OLIVER, G., SIDELL, N., FISKE, W., HEINZMANN, C., MOHANDAS, T., SPARKES, R. S., and DEROBERTIS, E. M. (1989). Complementary homeo protein gradients in developing limb buds. Genes Den 3, 641650. OLIVER, G., WRIGHT, C. V. E., HARDWICKE, J., and DEROBERTIS, E. M. (1988). A gradient of homeo domain protein in developing forelimbs of Xenopus and mouse embryos. Cell 55, 1017-1024. SAUNDERS, J. W., JR., and GASSELING, M. T. (1968). Ectodermal-mesenchymal interactions in the origin of limb symmetry. 1n “Epithelial-Mesenchymal Interactions” (R. Fleischmajer and R. E. Billingham, Eds.), pp. 1-24. Williams and Wilkins, Baltimore, MD.
535
TICKLE, C., ALBERTS, B. M., WOLPERT, L., and LEE, J. (1982). Local application of retinoic acid to the limb bud mimics the action of the polarizing region. Nature 296, 564-565. TICKLE, C., LEE, J., and EICHELE, G. (1985). A quantitative analysis of the effect of all-trans-retinoic acid on the pattern of chick wing development. Dev. Biol. 109, 82-95. TICKLE, C., SUMMERBELL, D., and WOLPERT, L. (1975). Positional signailing and specification of digits in chick limb morphogenesis. Nafur-e 254, 199-202. THALLER, C., and EICHELE, G. (1987). Identification and spatial distribution of retinoids in the developing chick limb bud. Nature 327, 625-628. WANEK, N., GARDINER, D. M., MUNEOKA, K., and BRYANT, S. V. (1991). Conversion by retinoic acid of anterior cells into ZPA cells in the chick wing bud. Nuturf 350,81-83.
BIOLOGY
148, 529-535
(1991)
along the AnteriorA Gradient of Gap Junctional Communication Posterior Axis of the Developing Chick Limb Bud CAROLINEN. Department
qf Anatomy,
Uni?lersity
D. COELHOANDROBERTAKOSHER of Connecticut Accepted
Health
August
Center,
Farmingfcm,
Connecticut
06030
2.9, 19.91
A modification of the scrape-loading/dye transfer technique was used to study gap junctional communication along the anterior-posterior (A-P) axis of embryonic chick wing buds at an early stage of development (stage 20121) when positional values along the A-P axis are being specified. Extensive intercellular transfer of the gap junction-permeable dye, lucifer yellow, from scrape-loaded mesenchymal cells to contiguous cells occurs in the posterior mesenchymal tissue of the wing bud adjacent to the zone of polarizing activity, which is thought to be the source of a diffusible morphogen that specifies A-P positional identity according to its local concentration. Considerably less transfer of lucifer yellow dye occurs in scrape-loaded mesenchymal tissue in the middle of the limb bud compared to posterior mesenchymal tissue, and little or no transfer of lucifer yellow is observed in the mesenchymal tissue in the anterior portion of the limb bud. No intercellular transfer of the gap junction-impermeable dye, rhodamine dextran, occurs in any region of the limb bud. These results indicate that there is a gradient of gap junctional communication along the A-P axis of the developing chick wing bud. This gradient of gap junctional communication along the A-P axis might generate a graded distribution of a relatively low molecular weight intracellular regulatory molecule involved in specifying A-P positional identities. ,2’ 1991 Academic Press, Inr. INTRODUCTION
A problem of major importance in development concerns the cellular and molecular mechanisms involved in generating the three-dimensional spatial organization of the various morphologically distinct skeletal elements of the limb in their appropriate position and sequence along the anterior-posterior (A-P) axis. A small group of mesenchymal cells located at the posterior margin of the developing limb bud appear to play an important role in regulating pattern formation along the A-P axis. When cells from this posterior region, which is known as the zone of polarizing activity (ZPA), are transplanted to the anterior margin of a host limb bud, they elicit the formation of duplicated distal skeletal elements from the host tissue which exhibit mirror image symmetry to the normal elements (Saunders and Gasseling, 1968). Several observations suggest that the ZPA is the source of a diffusible morphogen which forms a concentration gradient across the A-P axis of the limb, and that high concentrations of the morphogen specify the formation of posterior structures, while progressively lower concentrations specify the formation of progressively more anterior structures (Tickle et al., 1975). Several recent studies have provided evidence indicating that retinoic acid may be the putative morphogen released by the ZPA (see Brickell and Tickle, 1989; Eichele, 1989, for reviews). Implantation of beads soaked in retinoic acid into the anterior margin of the limb bud provokes the same mirror-image duplication 529
of skeletal elements as does implantation of ZPA tissue (Tickle et ah, 1982,1985). Moreover, endogenous retinoic acid is distributed in a graded fashion across the A-P axis of the limb bud such that its concentration is 2.5fold higher in the ZPA-containing posterior quarter of the limb bud than in the anterior portion (Thaller and Eichele, 1987). Furthermore, nuclear receptors and cytoplasmic binding proteins for retinoic acid are present in the developing limb bud (Maden et aZ., 1988; Dolle et al., 1989b). It should be noted, however, that recent studies suggest that retinoic acid may function by establishing or inducing the formation of the ZPA, which in turn might produce another factor that more directly specifies A-P positional identities (Noji et al., 1991; Wanek et al., 1991). Ultrastructural studies have revealed the presence of gap junctions between limb mesenchymal cells at early stages of limb development (Kelley and Fallon, 1978, 1983). Furthermore, grafts composed of ZPA tissue and anterior limb mesenchymal cells that had been loaded with antibodies against rat liver gap junctional proteins are considerably impaired in their ability to elicit the formation of duplicated skeletal elements when implanted into the anterior margin of limb buds, suggesting the possibility that gap junctional communication may be involved in regulating pattern formation (Allen et cd., 1990). In the present investigation, we have studied gap junctional communication along the A-P axis of embryonic chick wing buds at an early stage of development in OOlZ-1606/91 Copyright All rights
$3.00
W 1991 by Academic Press, Inc. uf reproduction in any form reserved.
530
DEVELOPMENTALBIOLOGY V0~~~~148,1991
which positional values along the A-P axis are being specified. We have found that extensive gap junctional communication occurs among posterior mesenchymal cells of the limb bud that are adjacent to the ZPA, and that progressively less intercellular communication occurs among mesenchymal cells in progressively more anterior regions of the limb bud. This gradient of gap junctional communication along the A-P axis suggests that intercellular signalling via gap junctions may be involved in specification of pattern along the A-P axis of the developing limb. MATERIALS
AND
METHODS
Gap junctional communication was assayed by monitoring the intercellular transfer of lucifer yellow dye using a modification of the scrape-loading/dye transfer technique (El-Fouly et al, 1987). Wing buds were removed from stage 20/21 (Hamburger and Hamilton, 1951) White Leghorn chick embryos, and the limb mesenchyme at various locations along the A-P axis was incised perpendicular to the apical ectodermal ridge or to the base of the limb with a fine scalpel blade. The incised limb buds were then incubated in a 0.05% solution of the gap junction-permeable dye, lucifer yellow (Sigma) and the gap junction-impermeable dye, rhodamine dextran (Molecular Probes, Inc.) in phosphatebuffered saline (PBS). Transfer of lucifer yellow from the incised cells to contiguous cells via gap junctions was allowed to proceed for 2-20 min, after which the tissues were washed with PBS; fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4; washed with PBS; and examined by fluorescence microscopy either before or after being mounted under cover slips in 10% glycerin in PBS. In order to effectively and consistently observe differential gap junctional communication along the A-P axis, it is important that the incisions through the mesenthyme in different regions of the same limb bud be as uniform in depth as possible. In particular, the depth of the incisions should extend about one-half of the way through the mesenchyme, since inconsistent results are obtained when the incisions are too superficial, and excessive damage and distortion of the tissue occurs when the mesenchyme is completely sliced. The uniformity of
the incisions in the different regions of the limb mesenthyme can be judged by comparing the extent of loading of the gap junction-impermeable dye, rhodamine dextran, into the wounded cells at the edge of the incisions. Only specimens in which comparable loading of rhodamine dextran was observed were used to evaluate the extent of lucifer yellow dye transfer in the different regions of the limb mesenchyme. Of the hundreds of samples we have attempted, we estimate that we can obtain uniform incisions in the different limb regions at least 60% of the time, and when such incisions are achieved, the gradient of dye transfer is clearly evident at least 90% of the time. RESULTS
Gap junctional communication as assayed by the intercellular transfer of lucifer yellow dye was monitored at various locations along the A-P axis of stage 20/21 wing buds, a stage of development in which positional values along the A-P axis are being specified. As shown in Fig. 1, during a 2-min incubation period extensive intercellular transfer of lucifer yellow dye from scrapeloaded mesenchymal cells to contiguous cells occurs in the posterior mesenchymal tissue adjacent to the ZPA, indicating extensive gap junctional communication is occurring in the posterior portion of the limb bud. Considerably less transfer of lucifer yellow dye occurs in scrape-loaded mesenchymal tissue in the middle of the limb bud compared to posterior mesenchymal tissue (Fig. l), and little transfer of lucifer yellow is observed in the mesenchymal tissue in the anterior portion of the limb bud (Fig. 1). The distance traversed by the dye during the 2-min incubation period ranges from about 1.5to 2.5-fold greater in the posterior mesenchymal tissue than in the mesenchymal tissue in the middle of the limb and from about 3- to 12-fold greater than in the anterior mesenchymal tissue. A similar gradient of dye transfer along the A-P axis was observed when scrapeloaded limb buds were incubated in lucifer yellow for up to 20 min. As illustrated in Fig. 2, lucifer yellow dye is located intracellularly throughout the cells contiguous to the incision in posterior mesenchyme. This observation indicates that lucifer yellow is being transferred from cell to
FIG. 1. Fluorescence (A-F) and corresponding bright light (a-f) photomicrographs illustrating the extent of intercellular transfer of lucifer yellow dye via gap junctions among mesenchymal cells in various regions along the anterior-posterior axis of stage 20/21 embryonic chick wing buds. The posterior border of the limb buds is indicated by the letter P and the anterior border is indicated by the letter A. Note that extensive intercellular transfer of lucifer yellow from scrape-loaded mesenchymal cells to contiguous cells occurs in the posterior mesenchymal tissue that is adjacent to the zone of polarizing activity (ZPA) which is located at the posterior margin of the limb buds. Considerably less transfer of lucifer yellow occurs in scrape-loaded mesenchymal tissue in the middle of the limb buds, and little transfer of the dye occurs in the mesenchyma1 tissue in the anterior portion of the limb bud. x50.
532
DEVELOPMENTALBIOLOGY
V0~~~~148,1991
COELHOANDKOSHER
Grip Ju~fiot~s
cell, and that the differential pattern of dye transfer along the A-P axis reflects differences in gap junctional communication in the various limb regions. Furthermore, as shown in Figs. 3A and 3B, the gap junction-impermeable dye, rhodamine dextran, remains localized in cells at sites of incision and is not transferred to contiguous cells. As shown in Figs. 3C and 3D, when scrapeloaded limb buds were fixed within seconds after being exposed to lucifer yellow dye, comparable amounts of the dye were localized at the sites of incision in all regions of the limb bud, indicating that comparable amounts of the dye are initially loaded into the incised cells in the different regions of the limb mesenchyme. This observation indicates that there are not major differences in the extent of dye loading in the different limb regions and that loading occurs to a sufficient extent in a reasonably comparable number of cells to evaluate reliably the dramatic differences in the extent of lucifer yellow transfer to contiguous cells in the different regions of the limb bud. DISCUSSION
The results of the present study indicate that there is a gradient of gap junctional communication along the A-P axis of the developing chick wing bud, with extensive intercellular communication occurring among posterior mesenchymal cells adjacent to the ZPA and progressively less communication occurring among mesenchymal cells in progressively more anterior regions of the limb bud. The observed gradient of gap junctional communication along the A-P axis and its relationship to the ZPA suggests that intercellular signalling via gap junctions may be involved in specification of positional values along the A-P axis of the developing limb. A gradient of gap junctional communication along the A-P axis would be an excellent mechanism for generating a graded distribution of a relatively low molecular weight intracellular regulatory molecule involved in specifying A-P positional values. It can be argued that gap junctions that might be present among anterior mesenchymal cells might be shut down in response to damage elicited by scrape-loading, whereas the gap junctions among the posterior mesenchymal cells might not be shut down in response to the scrape-loading procedure. Although this possibility is difficult to conclusively eliminate, the fact that little or no lucifer yellow dye transfer is detectable among the
FIG. 2. High magnification fluorescence photomicrograph bud. Lucifer yellow dye is located intracellularly throughout from cell to cell.
trrd
Limb
Paffrrn
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anterior mesenchymal cells regardless of if the cells are incubated in the dye for a few seconds or up to 20 min argues against it. The fact that little or no transfer of lucifer yellow is detectable in the anterior mesenchyme when the cells are fixed within seconds after being exposed to the dye argues against the possibility that extremely rapid transfer and dissipation of the dye is occurring among the anterior mesenchymal cells. The gradient of gap junctional communication along the A-P axis of the limb bud that we have observed combined with the studies of Allen et al. (1990) provides strong incentive for considering and further investigating the role of gap junctional signalling in the regulation of pattern formation along the A-P axis. Our observations are consistent with the suggestions that close range cell-cell interactions are involved in regulating positional identities (French et al., 1976; Bryant et al., 1986; Wanek et al., 1991). Considerable evidence has accumulated suggesting that retinoic acid may be the diffusible morphogen released by the ZPA that becomes distributed in a graded fashion across the A-P axis of the limb bud and specifies positional values along the axis (Brickell and Tickle, 1989; Eichele et aZ., 1989). It is therefore of considerable interest that retinoic acid is a potent modulator of gap junctional communication in several cell types (Hossain et al, 1989; Mehta et al., 1989). In particular, higher concentrations of retinoic acid enhance gap junctional communication, while lower concentrations of retinoic acid inhibit intercellular communication (Hossain et al., 1989; Mehta et al., 1989). It is therefore conceivable that the higher concentrations of retinoic acid present in the posterior region of the limb bud may promote the extensive gap junctional communication that is occurring in this region, while the lower concentrations of retinoic acid in the anterior region of the limb bud may be involved in inhibiting cell-cell communication. Retinoic acid may thus generate a gradient of gap junctional communication across the A-P axis that in turn establishes a gradient of a gap junction-permeable regulatory molecule that specifies positional values. This suggestion interfaces well with the recent studies indicating that retinoic acid might act by establishing or inducing the formation of the ZPA, which, in turn, might produce another factor that more directly specifies A-P positional identities (Noji el ah, 1991; Wanek et al., 1991). In addition to the gradients of gap junctional communication and retinoic acid, certain homeobox-containing
of scrape-loaded mesenchymal cells in the posterior region of a stage 20/21 wing the cells contiguous to the incision, indicating that the dye is being transferred
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tion and/or retinoic acid concentrations might regulate the differential pattern of expression of homeobox-containing genes, or vice versa, must be considered. All of these observations are consistent with the possibility that interacting opposing gradients of expression of various molecules may be involved in determining positional values along the A-P axis during limb development. This research was supported by NIH Grants HD22896 and HD22610 to R.A.K. REFERENCES
FIG. 3. (A and B) The posterior mesenchyme of a scrape-loaded stage 20/21 wing bud that had been incubated for 2 min in the presence of the gap junction-impermeable dye, rhodamine dextran (A) and the gap junction-permeable dye, lucifer yellow (B). Rhodamine dextran-labeled cells are limited to the site of incision (A), whereas lucifer yellow is transferred to contiguous cells (B). (C) A scrape-loaded stage 20/21 wing bud that was fixed within seconds after being exposed to lucifer yellow dye. Note that comparable amounts of the dye are localized at the sites of incision in all regions of the limb bud, indicating that comparable amounts of the dye are initially loaded into the incised cells in the different regions of the limb bud mesenthyme. (D) A higher magnification photomicrograph illustrating that lucifer yellow is indeed loaded into incised cells in the anterior mesenthyme.
genes, which have been implicated in the regulation of positional identity during development, are also expressed in a graded fashion along the A-P axis of the developing limb bud. For example, the chicken homeobox-containing gene GHox-8 (Coelho et al, 1991) and the chicken cognate of the Xenopus XlHbox 1 gene (the homolog of mouse Hox-3.3) (Oliver et al., 1988) are expressed predominantly in the anterior compartment of the distal mesoderm of chick limb buds, whereas several members of the HOX-4 cluster of homeobox-containing genes exhibit the complementary pattern of expression, being predominantly expressed in the ZPA-containing posterior mesoderm of the limb bud (Dolle et ah, 1989a; Oliver et al., 1989; Nohno et al, 1991). Therefore, the possibility that differential gap junctional communica-
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