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.     

Retinoic acid, midkine, and defects of secondary neurulation

BACKGROUND: Retinoic acid (RA) is necessary for normal differentiation of the tail bud into the secondary neural tube. Excess RA, however, is teratogenic and causes neural tube defects (NTDs). The way in which RA modulates secondary neurulation is unclear but probably involves RA-regulated downstream genes such midkine (MK), which encodes a growth factor implicated in tail bud mesenchymal-neuroepithelial conversion. Our objective was to determine whether RA-deficiency would produce similar defects and if MK is involved. METHODS: Citral, a drug that blocks endogenous RA formation, as well as a neutralizing antibody, were used to block RA activity in chick embryos. Immunohistochemistry and in situ hybridization were used to localize RA and MK in the tail bud. Competitive RT-PCR was used to examine the effects of excess RA and RA deficiency due to citral on the expression of MK mRNA. RESULTS: Citral-induced NTDs displayed a morphological resemblance to those caused by excess RA. However, citral treatment did not significantly increase embryonic mortality, and RA rescue of citral-treated embryos proved unsuccessful. MK mRNA was detected in the differentiating tail bud by in situ hybridization. Competitive RT-PCR showed that excess RA decreased MK expression by 60%. Doses of citral that caused a comparable incidence of defects, however, caused only a 25% decrease. CONCLUSIONS: The results show that excess RA and RA deficiency both cause defects of secondary neurulation. While excess RA decreased MK expression, RA deficiency had minimal effects. However, whether or not MK is an intermediary in the developmental phenomena regulated physiologically or pathologically by RA remains to be elucidated.     

Microbubble-enhanced sonoporation: efficient gene transduction technique for chick embryos

The gene transduction technique is a useful method to study gene functions that underlie vertebrate embryogenesis. In this study, a new gene transduction technique is reported using microbubble-enhanced sonoporation (hereafter referred to as sonoporation) to achieve ectopic and transient gene expression for several embryonic organs including embryonic chick limb bud mesenchymes. The technique proposed in this study has the advantages of 1) relatively simple gene transduction procedures, and 2) efficient exogenous gene transduction and expression with lower damages to embryos. Green fluorescent protein (GFP) or LacZ was misexpressed in limb bud mesenchymes by sonoporation, with the introduced expression transiently detected in the injected sites. Most of the transduced chick embryos survived without showing significant embryonic abnormalities or cell death after sonoporation. To demonstrate its efficacy for assessing the effect of transient gene transduction, the Shh (sonic hedgehog) was transduced into the developing chick limb bud. The transduced limb bud displayed limb malformations including partial digit duplication. Advantages and possible future applications in relation to this method are discussed.     

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.     

CXCL14 expression during chick embryonic development

Chemokines are small secreted signalling molecules best known for their roles as chemoattractants for cells of the immune system. CXCL12 and its receptor CXCR4 comprise one chemokine signalling pathway with essential functions in non-immune cell types during embryonic development. CXCL14, a chemokine-encoding gene related to CXCL12, is developmentally regulated in zebrafish and Xenopus embryos, but its role during embryogenesis remains unknown. Here we describe the embryonic expression pattern of CXCL14 in an amniote, the chick. Although expression in some regions is conserved with that of fish and frog, chick CXCL14 displays a complex pattern of expression in several novel sites. We analyse the expression pattern in the branchial arches, trigeminal placode and ganglion, inner ear, dorsal midline of the brain, somites, trunk neural tube and limb bud. Expression in several domains raises the possibility that CXCL14 may be involved in some of the same developmental events during which CXCL12-CXCR4 signalling is known to play a role.     

The respecification of limb pattern by new synthetic retinoids and their interaction with cellular retinoic acid-binding protein

We describe here experiments to examine the role of cellular retinoic-acid-binding protein (CRABP) during the induction of limb duplication in the chick limb bud and regenerating axolotl limb by retinoids. A newly synthesised class of retinoic acid analogues have been used because among them, some have been specifically designed with the property of binding to the retinoic acid receptors, but not to CRABP. We can thus test whether binding to CRABP is an obligatory step during limb respecification. The binding of four of these compounds to chick limb bud and axolotl CRABP was tested in sucrose density gradient assays and then their potencies at inducing limb duplications tested. Two of the four compounds do not bind to limb CRABP and yet are able to induce limb duplications, suggesting that an interaction with CRABP is not an obligatory step in the process. However, the two compounds which do bind to CRABP are more potent than the two which do not, suggesting that an interaction with CRABP may, nevertheless, increase the potency of a retinoid.     

Teratogenic effects of retinoic acid and dimethylsulfoxide on embryonic chick wing and somite

In order to document the stage(s) at which the embryonic chick wing bud is sensitive to vitamin A teratogenesis and the kinds of defects produced by vitamin A insult to the embryonic chick wing, 1-microgram doses of retinoic acid (1 microliter RA in 90% DMSO at a concentration of 1 microgram/microliter) were locally applied to the right wing bud of chick embryos at stages 17-23 (Hamburger and Hamilton: J. Morphol., 88:49-92, '51), and the resulting limb skeleton anatomy was observed at 10 days of incubation. Local application of RA at stages 17-20 resulted in distal wing skeleton defects. There were significantly more wing skeleton defects among embryos treated at these stages with RA solution than among solvent (DMSO)-treated control embryos and than among untreated control embryos. Wings of embryos treated with RA at stages 21-23 were always normal. Scapular and vertebral defects were seen at 10 days of incubation among embryos which had been treated prior to stage 21 with both the RA solution and the solvent control. Statistical analysis and histological data suggest that scapular and vertebral defects were caused by DMSO-induced damage to somites.     

Isolation of the avian homologue of the homeobox gene Mox2 and analysis of its expression pattern in developing somites and limbs

We have isolated the cDNA of avian Mox2 and analyzed its expression pattern during somitogenesis and limb bud formation. Mox2 plays an important role in limb muscle differentiation in the mouse. Mox2 is expressed in the somites of developing chick embryos and in presumptive migrating myoblasts from the dermomyotome to the limb buds. It is also expressed in the ventral and dorsal part of limb buds and is associated with non-proliferating myoblasts. Significant differences were observed in chick and mouse expression patterns, namely in the chick dermomyotome and limb.     

Expression of the short stature homeobox gene Shox is restricted by proximal and distal signals in chick limb buds and affects the length of skeletal elements

SHOX is a homeobox-containing gene, highly conserved among species as diverse as fish, chicken and humans. SHOX gene mutations have been shown to cause idiopathic short stature and skeletal malformations frequently observed in human patients with Turner, Leri-Weill and Langer syndromes. We cloned the chicken orthologue of SHOX, studied its expression pattern and compared this with expression of the highly related Shox2. Shox is expressed in central regions of early chick limb buds and proximal two thirds of later limbs, whereas Shox2 is expressed more posteriorly in the proximal third of the limb bud. Shox expression is inhibited distally by signals from the apical ectodermal ridge, both Fgfs and Bmps, and proximally by retinoic acid signaling. We tested Shox functions by overexpression in embryos and micromass cultures. Shox-infected chick limbs had normal proximo-distal patterning but the length of skeletal elements was consistently increased. Primary chick limb bud cell cultures infected with Shox showed an initial increase in cartilage nodules but these did not enlarge. These results fit well with the proposed role of Shox in cartilage and bone differentiation and suggest chick embryos as a useful model to study further the role of Shox in limb development.     

The cellular retinoic acid binding proteins

The two cellular retinoic acid binding proteins, CRABP I and CRABP II, belong to a family of small cytosolic lipid binding proteins and are highly conserved during evolution. Both proteins are expressed during embryogenesis, particularly in the developing nervous system, craniofacial region and limb bud. CRABP I is also expressed in several adult tissues, however, in contrast, CRABP II expression appears to be limited to the skin. It is likely that these proteins serve as regulators in the transport and metabolism of retinoic acid in the developing embryo and throughout adult life. It has been proposed that CRABP I sequesters retinoic acid in the cytoplasm and prevents nuclear uptake of retinoic acid. A role in catabolism of retinoic acid has also been proposed. Recent gene targeting experiments have shown that neither of the two CRABPs are essential for normal embryonic development or adult life. Examination of CRABP I expression at subcellular resolution reveals a differential cytoplasmic and/or nuclear localization of the protein. A regulated nuclear uptake of CRABP I implies a role for this protein in the intracellular transport of retinoic acid. A protein mediated mechanism which controls the nuclear uptake of retinoic acid may play an important role in the transactivation of the nuclear retinoic acid receptors.     

Position specific binding of a monoclonal antibody in chick limb buds

To analyze the molecular mechanism of the limb pattern formation, we have tried to make monoclonal antibodies against antigens from chick limb buds. We obtained one antibody named AV-1 which recognized a specific region of chick limb buds. AV-1 reacted with the distal portion of the anteroventral mesoderm of only developmentally early chick limb buds. Grafts of ZPA region tissue to an anterior site in an embryonic chick wing bud resulted in mirror-image dupliction of the AV-1 antigen region. These data show the possibility that this antigen plays some role in the limb pattern formation. This is the first evidence that a position specific substance really exists in developmentally early limb buds in which the pattern has been considered to be unspecified.     

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.