Effects of localized application of transforming growth factor beta 1 on developing chick limbs

The effects of exogenous transforming growth factor beta (TGF-beta) on chick limb development in vivo were studied by implanting carriers of TGF-beta 1 into developing wing buds. Agarose beads were soaked in solutions containing TGF-beta 1 and implanted into wing buds at stages 18 to 27. Localized application of TGF-beta 1 to distal regions of the wing bud caused specific skeletal elements in the limb to be reduced or absent. The particular proximal-distal limb element affected depended on the stage at which the bead was implanted. Position of the bead in the anterior-posterior axis also influenced the pattern of affected structures. Experiments in which TGF-beta 1 beads were implanted and then removed at 24- and 48-hr intervals indicate that there are specific periods during which a skeletal element appears to be sensitive to the effects of exogenous TGF-beta 1. In a few cases, beads placed in proximal positions in later staged limbs resulted in formation of ectopic cartilage near the bead. These results suggest that exposure to exogenous TGF-beta 1 in vivo influences the development of skeletal elements in the chick limb in a stage- and position-dependent manner.     

Retinoic acid promotes proliferation and chondrogenesis in the distal mesodermal cells of chick limb bud

Distal and proximal mesoderm of chick limb bud was respectively dissociated and cultured in the medium containing various concentrations of retinoic acid (RA). At low concentrations (5-50 ng/ml), RA promoted proliferation and chondrogenesis in the distal mesodermal cells. The distal cells of stage 20-24 limb buds were responsive to RA, although those of stages 25-27 were unresponsive. Both the cells of anterior and posterior regions of the distal mesoderm were responsive to RA, while the cells of proximal mesoderm were unresponsive. At higher concentrations, the growth-promoting effect of RA was reduced and chondrogenesis in the distal cells was rather inhibited. These results were discussed in relation to the role of RA as the morphogen in normal limb development and experimental duplicate formation.     

Regulation and Function of SF/HGF during Migration of Limb Muscle Precursor Cells in Chicken

Limb muscles of vertebrates are derived from migratory dermomyotomal cells which emanate from a limited number of somites located adjacent to the developing limb buds. We have generated additional limb buds in chicken embryos by implantation of FGF-beads into the interlimb region in order to analyze whether these somites can be programmed to supply ectopic limbs with myogenic precursor cells. We show that migrating myogenic precursor cells are released from somites at the level of the newly formed limb, even when cell migration into the natural limb has been completed. The implantation of FGF beads in the lateral plate mesoderm rapidly induces SF/HGF expression. FGF beads implanted between HH stages 10 and 12 inhibit limb bud formation or shift the normal limb position. When an additional FGF bead was implanted at the original limb position at HH stage 15, SF/HGF expression was transiently induced to low levels without inducing a new limb. This demonstrates that the initial induction of SF/HGF by FGF does not require limb formation. Expression of SF/HGF during early limb bud stages was found in the entire developing bud and the adjacent lateral plate mesoderm with direct contacts to the lateral edge of the dermomyotome. Later, the SF/HGF expression domain retracts to a distal region below the apical ectodermal ridge. To investigate the role of SF/HGF in the migratory process, we implanted beads soaked in SF/HGF-alone or together with FGF into different locations of the developing chick embryo. In the experiments SF/HGF caused delamination of migratory cells from the dermomyotomal epithelium but no chemotactic attraction of migrating cells toward the SF/HGF source.     

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.     

Regulation of the proportion of insulin cells in embryonic chick pancreas: effect of a growth factor-reduced extracellular matrix in combination with retinoic acid

Both retinoic acid (RA) and transforming growth factor (TGF)-beta1 are known to be influential in the development of insulin cells. Respectively, they increase and decrease the proportion of insulin cells when added to cultures of embryonic chick dorsal pancreatic buds. The aim of this study was to define the action of RA in the presence of decreased levels of TGF-beta1, as are found in growth factor-reduced Matrigel (GFRM), on the proportion of insulin cells. The endodermal component of 5-d chick dorsal pancreatic buds was explanted on to GFRM. Retinoic acid (10(-6) M) was added to Ham's F12 culture medium containing insulin (5 microg/ml), transferrin (5 microg/ml), and selenium (10(-10) M) (F12.ITS). Control explants were cultured in F12.ITS alone or in F12.ITS containing dimethyl sulfoxide (DMSO). After 7 d in culture, insulin and glucagon cells were localized immunocytochemically; changes in numbers of insulin cells were expressed as a percentage of insulin plus glucagon cells. Medium containing RA or DMSO increased the proportion of insulin cells significantly compared with the proportion in the explants cultured in F12.ITS medium alone.     

Expression and regulation of chicken fibroblast growth factor homologous factor (FHF)-4 at the base of the developing limbs

Fibroblast growth factor homologous factors (FHFs) have been implicated in limb and nervous system development. In this paper we describe the expression of the cFHF-4 gene during early chicken development. cFHF-4 is expressed in the paraxial mesoderm, lateral ridge, and, most prominently, in the posterior-dorsal side of the base of each limb bud. The expression pattern of cFHF-4 at the base of the limbs is not altered by tissue grafts containing the zone of polarizing activity (ZPA), by implants of Shh-expressing cells, or by implants of beads containing retinoic acid, nor does it depend on the distal growth of the limb as it is not altered in limb buds that are surgically truncated. In three chicken mutants affecting limb patterning - talpid(2), limbless, and wingless - altered patterns of cFHF-4 expression are correlated with abnormal nerve plexus formation and altered patterns of limb bud innervation. Similarly, ectopic expression of cFHF-4 is correlated with a local induction of limb-like innervation patterns when beads containing FGF-2 are implanted in the flank. In these experiments, both ectopic innervation and ectopic expression of cFHF-4 in the flank were observed regardless of the size of the FGF-2-induced outgrowths. By contrast, ectopic expression of Shh and HoxD13 are seen only in the larger FGF-2-induced outgrowths. Taken together, these data suggest that cFHF-4 regulates or is coregulated with early events related to innervation at the base of the limbs.     

Genistein represses the induction of prostatic buds by testosterone]

Genistein, a phytoestrogen and a kind of endocrine disrupters, inhibits tyrosine-specific protein kinase activity of the epidermal growth factor (EGF) receptor. It is also effective both in the suppression of the prostatic cell proliferation and the prostate carcinogenesis. We have recently demonstrated that several growth factors, like EGF, transforming growth factor-alpha (TGF-alpha), or keratinocyte growth factor (KGF), can induce prostatic bud formation in the absence of androgen. The present study was performed to investigate whether genistein can suppress testosterone-induced prostatic bud formation. Urogenital sinuses of 16.5-day male rat fetuses were cultured organotypically for 5 days in a serum-free medium containing 10 or 100 ng/ml genistein and 50 ng/ml testosterone. The number and total volume of prostatic buds were analyzed by laser scanning microscopy and computerized. We found that genistein inhibits significantly testosterone-induced prostatic bud formation. In the presence of genistein, cell proliferation of the sinus epithelium was suppressed and the number of prostatic buds and total volume of the buds were reduced as compared with those in the sinuses cultured with testosterone alone. Genistein did not appear to cause necrosis of the sinus. These results support our hypothesis that growth factors like EGF secreted from the sinus mesenchyme activated by testosterone are involved in the induction and stimulation of growth of the prostatic buds.     

Mouse limb bud cells respond to retinoic acid in vitro with reduced growth.

Retinoic acid (RA) has dramatic effects on the pattern of developing and regenerating vertebrate limbs. These effects are considered to result from RA-induced changes in the positional identity of limb cells, and involve the formation of extra structures. Whether the growth required to form the supernumerary parts of the pattern is a primary effect of RA treatment or a secondary effect that follows after a change in positional identity is not at present known. In this paper we have investigated the effects of RA treatment on the growth of cells from anterior and posterior halves of mouse limb buds in vitro. We observed that under our culture conditions, limb bud cells treated with 1 nM to 1 microM RA (0.3 ng/ml to 300 ng/ml) continue to grow but do so at a significantly slower rate than control cultures. There is a maximum inhibition of growth (50% of controls) between 10 nM and 100 nM RA, which corresponds to the measured range of concentrations of RA in vivo. Our observation of a significant decrease in growth rate over a wide range of RA concentrations is consistent with comparable reports of growth inhibition for a large number of other cell types in vitro as well as with the observation that exogenous RA inhibits blastemal growth in amphibians during the period of exposure to RA. We propose that the effects of RA on growth, either enhancement in vivo or reduction in vitro, can be seen as consequences of the ability of RA to alter positional identity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stage-dependent responses of the developing lung to retinoic acid signaling

Morphological analysis of vitamin A-deficient rat fetuses and of retinoic acid receptor (RAR and RXR) mutant mice have demonstrated that retinoic acid (RA) is essential for lung development. To gainfurther insight into RA signaling pathways during primary lung budformation and lung branching, we have investigated the effects of RA and of a pan-RAR antagonist in cultures of whole embryos and lung explants. Treatment of E8.0 embryos with the pan-RAR antagonist inhibits the formation of the primitive respiratory system. On the other hand, treatment of E11.75 and E12.5 lung explants with RA inhibits branching morphogenesis, whereas treatment with the pan-RAR antagonist at the same developmental stages stimulates formation of distal buds. The inhibitory effect of RA on branching is strongly decreased in RARbeta null lungs, while enhancement of budding by the pan-RAR antagonist is not affected by an RARgamma null mutation. Additionally, cellular retinol binding protein one (CRBPI) null lungs are more sensitive than wild type lungs to the pan-RAR antagonist-induced stimulation of branching. These data indicate that retinoid signaling is indispensable for the formation of primary lung buds and the oesophagotracheal septum from the primitive foregut. They also suggest that at the pseudoglandular stage, RA signaling through RARbeta, but not RARgamma, inhibits distal bud formation thereby promoting the formation of conducting airways. Moreover, the level of CRBPI in the pseudoglandular lung appears to participate in the control of branching morphogenesis.     

The expression pattern of the chicken homeobox-containing gene GHox-7 in developing polydactylous limb buds suggests its involvement in apical ectodermal ridge-directed outgrowth of limb mesoderm and in programmed cell death

Abstract. The limb buds of the polydactylous mutant embryos, talpid ² and diplopodia -5, possess expanded distal apexes surmounted by prolongated thickened apical ectodermal ridges that promote the outgrowth and formation of digits from both the anterior and posterior mesoderm of the mutant limb buds. The chicken homeobox-containing gene GHox-7 exhibits an expanded domain of expression throughout the expanded subridge mesoderm of the mutant limb buds, providing support for the hypothesis that GHox-7 expression by subridge mesenchymal cells is involved in the outgrowth-promoting effect of the apical ectodermal ridge. During normal limb development GHox-7 is also expressed by the mesoderm in the proximal anterior nonchondrogenic periphery of the limb bud, which includes, but is not limited to the anterior necrotic zone. GHox-7 is also expressed in the posterior necrotic zone at the mid-proximal posterior edge of the limb bud. In contrast, GHox-7 is not expressed in either the proximal anterior or posterior peripheral mesoderm of talpid ² and diplopodia -5 limb buds which lack proximal anterior and posterior necrotic zones. Furthermore, retinoic acid-coated bead implants, which diminish cell death in the anterior necrotic zone, elicit a local inhibition of GHox-7 expression in the proximal anterior peripheral mesoderm. These results support the suggestion that GHox-7 may be involved in defining regions of programmed cell death during limb development. Furthermore, these studies indicate that the distal subridge and proximal anterior nonchondrogenic mesodermal domains of GHox-7 expression are independently regulated.     

Role of the chicken homeobox-containing genes GHox-4.6 and GHox-8 in the specification of positional identities during the development of normal and polydactylous chick limb buds.

During early stages of normal chick limb development, the homeobox-containing (HOX) gene GHox-4.6 is expressed throughout the posterior mesoderm of the wing bud from which most of the skeletal elements including the digits will develop, whereas GHox-8 is expressed in the anterior limb bud mesoderm which will not give rise to skeletal elements. In the present study, we have examined the expression of GHox-4.6 and GHox-8 in the wing buds of two polydactylous mutant chick embryos, diplopodia-5 and talpid2, from which supernumerary digits develop from anterior limb mesoderm, and have also examined the expression of these genes in response to polarizing zone grafts and retinoic acid-coated bead implants which induce the formation of supernumerary digits from anterior limb mesoderm. We have found that the formation of supernumerary digits from the anterior mesoderm in mutant and experimentally induced polydactylous limb buds is preceded by the ectopic expression of GHox-4.6 in the anterior mesoderm and the coincident suppression of GHox-8 expression in the anterior mesoderm. These observations suggest that the anterior mesoderm of the polydactylous limb buds is "posteriorized" and support the suggestion that GHox-8 and GHox-4.6, respectively, are involved in specifying the anterior non-skeletal and posterior digit-forming regions of the limb bud. Although the anterior mesodermal domain of GHox-8 expression is severely impaired in the mutant and experimentally induced polydactylous limb buds, this gene is expressed by the prolonged, thickened apical ectodermal ridges of the polydactylous limb buds that extend along the distal anterior as well as the distal posterior mesoderm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential expression of chicken CYP26 in anterior versus posterior limb bud in response to retinoic acid

Multiple studies indicate that quantitative control of the levels of all-trans-retinoic acid (RA) in the vertebrate embryo is necessary for correct development. The function of RA in cells is regulated by a number of coordinated mechanisms. One of those mechanisms involves controls on the rate of RA catabolism. Recently, enzymes capable of catabolizing RA were found to constitute a new family, called CYP26, within the cytochrome P450 superfamily. CYP26 homologues have been isolated from human, mouse, zebra fish, and recently from the chick. In this study, we examined the regulation of chicken CYP26 (cCYP26) expression by RA during the early phase of chick limb outgrowth. In the anterior limb mesenchyme and apical ectodermal ridge (AER), cCYP26 expression was induced in a concentration dependent manner by implanting beads soaked in 0.1, 1, and 5 mg/ml RA. The RA-induced expression of cCYP26 in anterior limb mesenchyme and the AER was detected as early as 1 hr after treatment and was not affected by the presence of cycloheximide. In contrast to the anterior limb, the induction of cCYP26 was dramatically reduced (or absent) when RA beads were implanted in the posterior limb mesenchyme. Furthermore, induction of cCYP26 expression in the anterior mesenchyme was inhibited by transplantations of the zone of polarizing activity (ZPA) and by Shh-soaked beads. Our data suggest that different mechanisms regulate retinoid homeostasis in the AER and mesenchyme during limb bud outgrowth. J. Exp. Zool. 290:136-147, 2001.     

Processes involved in retinoic acid production of small embryonic palatal shelves and limb defects

All-trans-retinoic acid (RA) is teratogenic to the embryonic mouse, producing malformations in many developing systems, including the limb bud and palate. High incidences of limb defects and cleft palate are induced at doses which are not maternally toxic and do not increase resorptions. Exposure to RA on gestational day (GD) 10 results in small palatal shelves, which fail to make contact on GD 14. The formation of small shelves could be a consequence of increased cell death, reduced proliferation, a combination of these effects, or some other effect such as inhibition of extracellular matrix production. After exposure to 100 mg RA/kg on GD 10, proliferation in mesenchymal cells of the palatal shelves was not reduced from GD 12 to GD 14 and the levels of cell death in control and treated shelves did not differ when observed by light and electron microscopy. The present study examines the effects of RA on cell death and proliferation from GDs 10-12 and compares the effects in palatal shelves and limb buds. Embryonic mice were exposed to RA suspended in corn oil (100 mg/kg on GD 10), a dose that was teratogenic but not maternally toxic or embryolethal. Embryos were collected at 4, 12, 24, 36, or 48 hr postexposure, and tissues which form the palate or limb were dissected from the embryos, stained by a modified Feulgen procedure, and whole mounted on slides. Mitotic index (MI) and percentage dead cells were determined for mesenchymal cells of the first visceral arch, maxillary process, or palatal shelf (depending on stage of development) and forelimb buds. In the palatal tissues from GD 10 to GD 12, RA did not significantly alter MI and percentage dead cells was significantly increased only at 4 hr postexposure. Some whole embryos were prepared for scanning electron microscopy (SEM). At 48 hr (GD 12) a reduction in the size of the shelves was not apparent on SEM. In the limb buds, RA did not increase percentage dead cells, but MI was significantly decreased. A decreasing rate of proliferation was detected in control facial tissues as development progressed, and this agrees with findings in rat and chick. Thus it appears that mesenchymal cell death and reduced proliferation are not responsible for the small palatal shelves seen on GD 14. RA did not increase cell death but inhibited proliferation in the limb bud, and this effect may contribute to the retarded development and malformations occurring in the limb.     

Plant regeneration via adventitious bud formation from cotyledon explants of Panax ginseng C. A. Meyer

Cotyledon explants of ginseng (Panax ginseng C. A. Meyer) produced somatic embryos directly on medium without growth regulators, with 89% of the explants forming somatic embryos. Cytokinin treatment greatly suppressed somatic embryo formation but stimulated the direct formation of adventitious buds. BAP treatment was more effective than the kinetin treatment for adventitious bud formation. Auxin (0.05 mg/l IBA) in combination with cytokinin enhanced adventitious bud formation, with the highest frequency, 40%, at 0.05 mg/l IBA and 5 mg/l BAP. Adventitious buds were mainly formed near the distal portion of the cotyledons, while somatic embryos were formed near the proximal excised margins. Shoots were developed from adventitious buds after transfer to MS medium with 10 mg/l GA₃. Root formation from the shoots was obtained after the shoots were transferred to half-strength MS medium with auxin (IAA). When the plants derived from adventitious buds were transferred to greenhouse soil, 36% were successfully acclimatized. Received: 7 November 1997 / Revision received: 12 January 1998 / Accepted: 7 February 1998     

Regulation of retinoic acid signaling during lung morphogenesis

Little is known about how retinoic acid (RA) synthesis, utilization and metabolism are regulated in the embryonic lung and how these activities relate to lung pattern formation. Here we report that early lung bud formation and subsequent branching morphogenesis are characterized by distinct stages of RA signaling. At the onset of lung development RA signaling is ubiquitously activated in primary buds, as shown by expression of the major RA-synthesizing enzyme, RALDH-2 and activation of a RARE-lacZ transgene. Nevertheless, further airway branching appears to require downregulation of RA pathways by decreased synthesis, increased RA degradation in the epithelium via P450RAI-mediated metabolism, and inhibition of RA signaling in the mesenchyme by COUPTF-II expression. These mechanisms controlling local RA signaling may be critical for normal branching, since we show that manipulating RA levels in vitro to maintain RA signaling activated as in the initial stage, leads to an immature lung phenotype characterized by failure to form typical distal buds. We show that this phenotype likely results from RA interfering with the establishment of a distal signaling center, altering levels and distribution of Fgf10 and Bmp4, genes that are essential for distal lung formation. Furthermore, RA upregulates P450RAI expression, suggesting the presence of feedback mechanisms controlling RA availability. Our study illustrates the importance of regional mechanisms that control RA availability and utilization for correct expression of pattern regulators and normal morphogenesis during lung development.     

Effects of Exogenously Applied Gibberellins and Thidiazuron on Phytohormone Profiles of Long-Shoot Buds and Cone Gender Determination in Lodgepole Pine

In long-shoot buds of lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia Engelm), cone-bud initiation and gender differentiation occur in a site-specific manner: female cone buds are normally restricted to the distal portion, whereas male cone buds are located in the proximal portion. Application of a paste containing two plant growth regulators gibberellins A₄ + A₇ (GA_4/7) combined with thidiazuron (TDZ) to long-shoot buds prior to cone-bud gender determination altered endogenous phytohormone profiles and induced female cone-bud formation in the proximal portion of the long-shoot bud, where male cone buds normally occur. Induced cone clusters observed in the following spring were either entirely female or a mixture of both female and male cones. Endogenous phytohormones in the long-shoot bud tissues were quantified by the stable isotope dilution method using high performance liquid chromatography-electrospray ionization tandem mass spectrometry in multiple-reaction monitoring mode. Applied GA_4/7 + TDZ led to increased concentrations of endogenous zeatin-type cytokinins, that is, trans-zeatin riboside and dihydrozeatin riboside, whereas concentrations of abscisic acid (ABA) and its catabolite, ABA glucose ester, were decreased, all relative to control, in untreated long-shoot bud tissue. Concentrations of extractable GA₄ and GA₇ declined in long-shoot bud tissues over 4 weeks following treatment with exogenous GA_4/7. This study demonstrates that high levels of endogenous zeatin-type cytokinins, together with reduced levels of ABA, both induced by applied GA_4/7 + TDZ, are positively associated with an increased female cone-bud formation in long-shoot buds.     

Expression of a homeobox gene in the chick wing bud following application of retinoic acid and grafts of polarizing region tissue.

Homeobox gene XlHbox 1 is expressed in a mesodermal gradient in vertebrate forelimbs with maximal expression anteriorly and proximally and may encode positional values. In chick wing buds, anterior cells can be reprogrammed to form posterior structures by grafts of polarizing region tissue and by beads soaked in retinoic acid (RA), which is a good candidate for an endogenous morphogen. Applications of RA anteriorly or at the bud apex, treatments which produce duplicated digits or truncations respectively, substantially increase the extent of mesodermal XlHbox 1 expression. Polarizing region grafts that also produce additional digits lead to a moderate increase. The effects of RA application and the behaviour of transplanted tissue show that only anterior cells are competent to express XlHbox 1 and that expression is cell autonomous. Ectodermal expression in wing buds is enhanced by RA but not by polarizing region grafts and ectoderm/mesoderm recombinations show that the mesoderm is irreversibly affected. The changes in mesodermal expression do not fit the predictions of the simple model that XlHbox 1 encodes anterior positional values but are correlated with a series of novel malformations of the shoulder girdle which, in normal wing buds, is derived from cells expressing XlHbox 1.     

Elevations in the endogenous levels of the putative morphogen retinoic acid in embryonic mouse limb-buds associated with limb dysmorphogenesis

Retinoic acid, an endogenous metabolite of vitamin A (retinol), possesses striking biological activity akin to a morphogen in developing and regenerating vertebrate limbs. Systemic administration of retinoic acid (RA) to pregnant mammals during the period of limb organogenesis invariably results in dose-dependent dysmorphogenesis. In an attempt to uncover the mode of action of RA in the developing limb bud we analyzed, by HPLC methods, the levels of RA and its metabolic precursor, retinol, in embryonic mouse tissues prior to and following maternal exposure to a teratogenic dose of RA. Detectable levels of both RA and its isomer 13-cis-retinoic acid were found in the limb buds of Day 11 mouse embryos (40 +/- 2 somites). Although retinol was the major retinoid found in ethanolic extracts of either whole embryo or the limb buds, the latter is enriched in RA compared to the whole embryo. This indicated either a higher degree of retinol metabolism or a sequestration of RA in the limb bud compared to the rest of the embryo at this stage of development. A study of the time course of retinoid levels in treated embryos showed that changes occur rapidly, are stable for several hours, and then begin to return to pretreatment levels. After a maternal dose of 10 mg/kg RA, which resulted in a mild degree of limb anomalies, peak RA levels in the limb bud increased 50-fold over the endogenous level; a full 300-fold increase was found after a 100 mg/kg dose which results in 100% incidence of phocomelia. Interestingly, a dose-dependent depression in retinol levels was observed after RA treatment both in maternal plasma as well as the embryo. Studies are in progress to trace the intracellular disposition of both retinol and RA as well as any further active metabolite of RA in the limb buds and other embryonic tissues.     

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.