Expression In Limb Buds Research Articles

Segmentation and limb formation during naupliar development of Tigriopus californicus (Copepoda, Harpacticoida)

Naupliar development in copepods includes the generation of usually five pairs of post-mandibular segments. Since copepod nauplii show no outer body articulation, the only indication of larval segmentation is the expression of limb buds. Yet, in copepods the timing and sequence of limb bud expression in larval development varies to a large degree. In harpacticoid nauplii for instance, the 1st maxillae are formed at an early naupliar stage. By contrast, the four remaining pairs of limb buds frequently appear simultaneously with the last naupliar stage. The complete process of larval segment formation takes place under the body surface and has never been described in detail. To broaden our knowledge of early segmentation in copepods, we here describe the segmentation of the harpacticoid nauplius Tigriopus californicus by analysing the expression of the segment marker Engrailed and uncover the sequential addition of seven post-mandibular segments. The stripe formation and arrangement of labelled cells corresponds largely to those of other crustaceans studied in this respect. Together with a morphological approach using histology, SEM, and 3D-reconstructions based on CLSM we solve the so far controversial identity of the external limb buds in the final naupliar stage. In contrast to previous studies, we can show that all limb pairs from the 1st maxillae to the 3rd thoracopods are formed. Yet, the anlage of the maxilliped (1st thoracopod) remains hidden underneath the cuticle being never externally expressed in the nauplius.

HOX-mediated LMO2 expression in embryonic mesoderm is recapitulated in acute leukaemias

The Lim Domain Only 2 (LMO2) leukaemia oncogene encodes an LIM domain transcriptional cofactor required for early haematopoiesis. During embryogenesis, LMO2 is also expressed in developing tail and limb buds, an expression pattern we now show to be recapitulated in transgenic mice by an enhancer in LMO2 intron 4. Limb bud expression depended on a cluster of HOX binding sites, while posterior tail expression required the HOX sites and two E-boxes. Given the importance of both LMO2 and HOX genes in acute leukaemias, we further demonstrated that the regulatory hierarchy of HOX control of LMO2 is activated in leukaemia mouse models as well as in patient samples. Moreover, Lmo2 knock-down impaired the growth of leukaemic cells, and high LMO2 expression at diagnosis correlated with poor survival in cytogenetically normal AML patients. Taken together, these results establish a regulatory hierarchy of HOX control of LMO2 in normal development, which can be resurrected during leukaemia development. Redeployment of embryonic regulatory hierarchies in an aberrant context is likely to be relevant in human pathologies beyond the specific example of ectopic activation of LMO2.

The molecular basis of human congenital limb malformations

This review focuses predominantly on the human congenital malformations caused by alterations affecting the morphoregulatory gene networks that control early limb bud patterning and outgrowth. Limb defects are among the most frequent congenital malformations in humans that are caused by genetic mutations or teratogenic effects resulting either in abnormal, loss of, or additional skeletal elements. Spontaneous and engineered mouse models have been used to identify and study the molecular alterations and disrupted gene networks that underlie human congenital limb malformations. More recently, mouse genetics has begun to reveal the alterations that affect the often-large cis-regulatory landscapes that control gene expression in limb buds and cause devastating effects on limb bud development. These findings have paved the way to identifying mutations in cis-regulatory regions as causal to an increasing number of congenital limb malformations in humans. In these cases, no mutations in the coding region of a presumed candidate were previously detected. This review highlights how the current understanding of the molecular gene networks and interactions that control mouse limb bud development provides insight into the etiology of human congenital limb malformations.

Opposing Functions of the ETS Factor Family Define Shh Spatial Expression in Limb Buds and Underlie Polydactyly

SummarySonic hedgehog (Shh) expression during limb development is crucial for specifying the identity and number of digits. The spatial pattern of Shh expression is restricted to a region called the zone of polarizing activity (ZPA), and this expression is controlled from a long distance by the cis-regulator ZRS. Here, members of two groups of ETS transcription factors are shown to act directly at the ZRS mediating a differential effect on Shh, defining its spatial expression pattern. Occupancy at multiple GABPα/ETS1 sites regulates the position of the ZPA boundary, whereas ETV4/ETV5 binding restricts expression outside the ZPA. The ETS gene family is therefore attributed with specifying the boundaries of the classical ZPA. Two point mutations within the ZRS change the profile of ETS binding and activate Shh expression at an ectopic site in the limb bud. These molecular changes define a pathogenetic mechanism that leads to preaxial polydactyly (PPD).

Conserved cis-regulatory regions in a large genomic landscape control SHH and BMP-regulated Gremlin1expression in mouse limb buds

BackgroundMouse limb bud is a prime model to study the regulatory interactions that control vertebrate organogenesis. Major aspects of limb bud development are controlled by feedback loops that define a self-regulatory signalling system. The SHH/GREM1/AER-FGF feedback loop forms the core of this signalling system that operates between the posterior mesenchymal organiser and the ectodermal signalling centre. The BMP antagonist Gremlin1 (GREM1) is a critical node in this system, whose dynamic expression is controlled by BMP, SHH, and FGF signalling and key to normal progression of limb bud development. Previous analysis identified a distant cis-regulatory landscape within the neighbouring Formin1 (Fmn1) locus that is required for Grem1 expression, reminiscent of the genomic landscapes controlling HoxD and Shh expression in limb buds.ResultsThree highly conserved regions (HMCO1-3) were identified within the previously defined critical genomic region and tested for their ability to regulate Grem1 expression in mouse limb buds. Using a combination of BAC and conventional transgenic approaches, a 9 kb region located ~70 kb downstream of the Grem1 transcription unit was identified. This region, termed Grem1 Regulatory Sequence 1 (GRS1), is able to recapitulate major aspects of Grem1 expression, as it drives expression of a LacZ reporter into the posterior and, to a lesser extent, in the distal-anterior mesenchyme. Crossing the GRS1 transgene into embryos with alterations in the SHH and BMP pathways established that GRS1 depends on SHH and is modulated by BMP signalling, i.e. integrates inputs from these pathways. Chromatin immunoprecipitation revealed interaction of endogenous GLI3 proteins with the core cis-regulatory elements in the GRS1 region. As GLI3 is a mediator of SHH signal transduction, these results indicated that SHH directly controls Grem1 expression through the GRS1 region. Finally, all cis-regulatory regions within the Grem1 genomic landscape locate to the DNAse I hypersensitive sites identified in this genomic region by the ENCODE consortium.ConclusionsThis study establishes that distant cis-regulatory regions scattered through a larger genomic landscape control the highly dynamic expression of Grem1, which is key to normal progression of mouse limb bud development.

MicroRNA-221 Regulates Chondrogenic Differentiation through Promoting Proteosomal Degradation of Slug by Targeting Mdm2

MicroRNAs (miRNAs) are small RNAs that fulfill diverse functions by negatively regulating gene expression. Here, we investigated the involvement of miRNAs in the chondrogenic differentiation of chick limb mesenchymal cells and found that the expression of miR-221 increased upon chondrogenic inhibition. Blockade of miR-221 via peanut agglutinin-based antisense oligonucleotides reversed the chondro-inhibitory actions of a JNK inhibitor on the proliferation and migration of chondrogenic progenitors as well as the formation of precartilage condensations. We determined that mdm2 is a relevant target of miR-221 during chondrogenesis. miR-221 was necessary and sufficient to down-regulate Mdm2 expression, and this down-modulation of Mdm2 by miR-221 prevented the degradation of (and consequently up-regulated) the Slug protein, which negatively regulates the proliferation of chondroprogenitors. These results indicate that miR-221 contributes to the regulation of cell proliferation by negatively regulating Mdm2 and thereby inhibiting Slug degradation during the chondrogenesis of chick limb mesenchymal cells.

Fly Fishing with a Hedgehog

DEVELOPMENT The complexity of gene regulation mechanisms is also evident in the analysis by Amano et al. of the temporal and spatial patterns of Sonic hedgehog ( Shh ) expression. Shh is required for proper limb formation in mice. In the limb bud, it is expressed exclusively in a group of posterior mesenchymal cells called the zone of polarizing activity (ZPA). The authors found that colocalization of the Shh coding region and the long-range enhancer of its expression in limb bud, MFCS1, frequently occurs in the ZPA of day-10.5 limb buds but not by day 12.5, when Shh has been turned off. Chromosome conformation capture assays confirmed that MFCS1 and Shh interact directly in the limb bud. This interaction appears to indicate transcriptional competence; for active transcription to occur, a chromosome loop involving Shh needs to form, and evidence of loop formation was seen only in the ZPA. Such loops have been observed in other systems, and the authors propose that the interaction of MFCS1 and Shh promotes a looping-out of the chromosome that renders Shh accessible to transcription factors. This change in chromosomal configuration was observed in only a fraction of the ZPA cells at any one time; the resultant intermittent exposure of Shh may ensure proper control of levels of Shh needed for limb morphogenesis. — BJ Dev. Cell 16 , 10.1016/j.devcel.2008.11.011 (2009).

Expression of the NET family member Zfp503 is regulated by hedgehog and BMP signaling in the limb

The NET/Nlz family of zinc finger transcription factors contribute to aspects of developmental growth and patterning across evolutionarily diverse species. To date, however, these molecules remain largely uncharacterized in mouse and chick. We previously reported that limb bud expression of Zfp503, the mouse orthologue of zebrafish nlz2/znf503, is dependent on Gli3. Here, we show that Zfp503/Znf503 is expressed in a restricted pattern during mouse and chick embryogenesis, with particularly dynamic expression in the developing limbs, face, somites, and brain. We also add to our previous data on Gli3 regulation by showing that the anterior domain of Zfp503 expression in the mouse limb is responsive to genetic and nongenetic manipulation of hedgehog signaling. Finally, we demonstrate that posterior expression of Znf503 in the chick limb is responsive to bone morphogenetic protein (BMP) signaling, indicating that Zfp503/Znf503 may act at the nexus of multiple signaling pathways in development.

Analysis of paralogous pontin and reptin gene expression during mouse development

Evolutionarily conserved from yeast to human, the paralogous DNA helicases Pontin (Pont) and Reptin (Rept) are simultaneously recruited in multi-protein chromatin complexes that function in different aspects of DNA metabolism (transcription, replication and repair). When assayed, the two proteins were found to be essential for viability and to play antagonistic roles, suggesting that the balance of Pont/Rept regulates epigenetic programmes critical for development. Consistent with this, the two helicases are provided in the same embryonic territories during Drosophila development. In Xenopus, while transcribed in the same regions early in embryogenesis, pont and rept adopt significantly different patterns afterwards. Here we report that the two genes follow highly resembling transcription patterns in mouse embryos, with prominent expression in limb buds and branchial arches, organs undergoing mesenchymal-epithelial interactions and in motoneurones from cranial and spinal regions. Thus, simultaneous expression during development appears to constitute another feature of the evolutionary conservation of pont and rept genes.

Candidate downstream regulated genes of HOX group 13 transcription factors with and without monomeric DNA binding capability

Hox genes encode transcription factors that regulate the morphogenesis of developing embryos. In mammals, knowledge of the genetic pathways, including the possible direct or indirect targets, regulated by HOX proteins is extremely limited. To identify the downstream genes regulated by posterior HOX proteins, we expressed HOXA13 in mouse embryonic fibroblasts lacking paralog group 13 expression using a bicistronic HOXA13/EGFP retroviral vector. Microarray analysis identified 68 genes with significant, reproducible RNA expression changes (50 activated; 18 repressed) in stable HOXA13-expressing cells. Genes with the GO annotation terms “extracellular matrix” and “basement membrane” were greatly overrepresented, and several were shown to be regulated by HOX proteins in other studies. Among the genes strongly activated by HOXA13 were Enpp2, a bifunctional enzyme known to modulate tumor and normal cell motility and which is expressed in precartilaginous condensations; Fhl1, a transcription factor implicated in muscle cell differentiation and development; and M32486, a putative integral membrane molecule expressed in the female reproductive tract. Expression differences in the HOXA13-expressing cells were confirmed for selected downstream genes using semi-quantitative RT-PCR, and in vivo coexpression with Hoxa13 in the limb interdigital mesenchyme was demonstrated for many. For two candidates, Igfbp4 and Fstl, interdigital limb bud expression was reduced in Hoxa13 mutants. To explore whether paralogous and nonparalogous HOX proteins could regulate the same genes, we created new HOX cell lines and examined the expression of selected genes identified by the HOXA13 screen. HOXD13 similarly activated/repressed 6 tested candidates, demonstrating that multiple downstream genetic pathways may be regulated by paralog HOX proteins. In contrast, HOXA9 was only able to repress expression of some gene targets. A HOXD13 mutant, HOXD13 IQN > AAA, incapable of monomeric DNA-binding, activated the expression of 5 HOXA13-upregulated genes; but was incapable of repressing the expression of Ngef and Casp8ap2. Our results suggest that HOX protein–protein interactions without direct HOX DNA-binding may play a larger role in HOX transcriptional regulation than generally assumed, and DNA-binding appears critical for repression.

Elimination of a long-range cis-regulatory module causes complete loss of limb-specificShhexpression and truncation of the mouse limb

Mutations in a conserved non-coding region in intron 5 of the Lmbr1 locus, which is 1 Mb away from the sonic hedgehog (Shh) coding sequence, are responsible for mouse and human preaxial polydactyly with mirror-image digit duplications. In the mouse mutants, ectopic Shh expression is observed in the anterior mesenchyme of limb buds. Furthermore, a transgenic reporter gene flanked with this conserved non-coding region shows normal polarized expression in mouse limb buds. This conserved sequence has therefore been proposed to act as a long-range, cis-acting regulator of limb-specific Shh expression. Previous phylogenetic studies have also shown that this sequence is highly conserved among tetrapods, and even in teleost fishes. Paired fins of teleost fishes and tetrapod limbs have evolved from common ancestral appendages, and polarized Shh expression is commonly observed in fins. In this study, we first show that this conserved sequence motif is also physically linked to the Shh coding sequence in a teleost fish, the medaka, by homology search of a newly available genomic sequence database. Next, we show that deletion of this conserved intronic sequence by targeted mutation in the mouse results in a complete loss of Shh expression in the limb bud and degeneration of skeletal elements distal to the stylopod/zygopod junction. This sequence contains a major limb-specific Shh enhancer that is necessary for distal limb development. These results suggest that the conserved intronic sequence evolved in a common ancestor of fishes and tetrapods to control fin and limb development.

The core enhancer is essential for proper timing of MyoD activation in limb buds and branchial arches

Transgenic analyses have defined two transcriptional enhancers that regulate MyoD expression in mammals, the core enhancer and distal regulatory region; these enhancers exhibit complementary activities and together are sufficient to recapitulate MyoD expression in developing and mature skeletal muscle. The core enhancer is activated in presumptive muscle cells and determined myoblasts, suggesting an important role in initiating MyoD expression. Here, targeted mutagenesis in the mouse is used to identify necessary and redundant core enhancer functions. The core enhancer is essential for the timely initiation of MyoD expression in limb buds and branchial arches, as enhancer deletion delayed MyoD activation by 1 to 2 days in these muscle lineages. Functionally, this delay in MyoD transcription delayed the onset of muscle differentiation, as assayed by expression of the gene encoding for the early differentiation marker, Myogenin. In addition to these lineage-specific defects, a generalized, modest reduction in MyoD expression was observed in all muscle lineages and at all embryonic stages examined. Interestingly, however, a specific defect was not observed in the nascent myocytes at the medial and lateral aspects of the myotome, suggesting the existence of at least one other enhancer with this specificity. The core enhancer was also dispensable for Myf-5- and Pax-3-dependent regulation of MyoD transcription. These data demonstrate a differential requirement for core enhancer activity in muscle lineages derived from migratory precursors and suggest redundancy in cis regulatory mechanisms controlling myotomal MyoD expression.

Regulation of Tbx3 Expression by Anteroposterior Signalling in Vertebrate Limb Development

Tbx3 , a T-box gene family member related to the Drosophila gene optomotor blind ( omb ) and encoding a transcription factor, is expressed in anterior and posterior stripes in developing chick limb buds. Tbx3 haploinsufficiency has been linked with the human condition ulnar–mammary syndrome, in which predominantly posterior defects occur in the upper limb. Omb is expressed in Drosophila wing development in response to a signalling cascade involving Hedgehog and Dpp. Homologous vertebrate signals Sonic hedgehog (Shh) and bone morphogenetic protein 2 (Bmp2) are associated in chick limbs with signalling of the polarising region which controls anteroposterior pattern. Here we carried out tissue transplantations, grafted beads soaked in Shh, Bmps, and Noggin in chick limb buds, and analysed Tbx3 expression. We also investigated Tbx3 expression in limb buds of chicken and mouse mutants and retinoid-deficient quail in which anteroposterior patterning is abnormal. We show that Tbx3 expression in anterior and posterior stripes is regulated differently. Posterior Tbx3 expression is stable and depends on the signalling cascade centred on the polarising region involving Shh and Bmps, while anterior Tbx3 expression is labile and depends on the balance between positive Bmp signals, produced anteriorly, and negative Shh signals, produced posteriorly. Our results are consistent with the idea that posterior Tbx3 expression is involved in specifying digit pattern and thus provides an explanation for the posterior defects in human patients. Anterior Tbx3 expression appears to be related to the width of limb bud, which determines digit number.

Regulation of Tbx3 Expression by Anteroposterior Signalling in Vertebrate Limb Development

Tbx3, a T-box gene family member related to the Drosophila gene optomotor blind ( omb) and encoding a transcription factor, is expressed in anterior and posterior stripes in developing chick limb buds. Tbx3 haploinsufficiency has been linked with the human condition ulnar–mammary syndrome, in which predominantly posterior defects occur in the upper limb. Omb is expressed in Drosophila wing development in response to a signalling cascade involving Hedgehog and Dpp. Homologous vertebrate signals Sonic hedgehog (Shh) and bone morphogenetic protein 2 (Bmp2) are associated in chick limbs with signalling of the polarising region which controls anteroposterior pattern. Here we carried out tissue transplantations, grafted beads soaked in Shh, Bmps, and Noggin in chick limb buds, and analysed Tbx3 expression. We also investigated Tbx3 expression in limb buds of chicken and mouse mutants and retinoid-deficient quail in which anteroposterior patterning is abnormal. We show that Tbx3 expression in anterior and posterior stripes is regulated differently. Posterior Tbx3 expression is stable and depends on the signalling cascade centred on the polarising region involving Shh and Bmps, while anterior Tbx3 expression is labile and depends on the balance between positive Bmp signals, produced anteriorly, and negative Shh signals, produced posteriorly. Our results are consistent with the idea that posterior Tbx3 expression is involved in specifying digit pattern and thus provides an explanation for the posterior defects in human patients. Anterior Tbx3 expression appears to be related to the width of limb bud, which determines digit number.

Characterisation of hoxa gene expression in the chick limb bud in response to FGF.

We tested a diffusion gradient model for setting up overlapping domains of Hoxa gene expression in the chick limb bud. The model is based on morphogen production at the limb bud tip where the apical ridge is located and assumes that cells respond to a series of concentration thresholds. Consistent with the model, Hoxa13 gene expression rapidly switches off when the ridge is removed from stage 21/22 buds, while Hoxa11 and Hoxa10 expression is stable; Hoxa13 expression can be initiated and maintained in absence of the ridge by FGF soaked beads; the Hoxa13 domain first expands quickly and then slows up and the size is related to the dose of FGF4. Contrary to the model, addition of FGF4 to early limb buds does not activate Hoxa13 prematurely nor extend the Hoxa13 expression domain proximally. Therefore FGF4 signalling is necessary but not sufficient for Hoxa gene expression in the limb bud.

Inhibitory action of BMPs on Pax1 expression and on shoulder girdle formation during limb development.

Pax1 expression in vertebrate limb buds is confined to cells in a discrete anterior proximal domain (Timmons et al. [1994] Development 120:2773-2785; Ebensperger et al. [1995] Anat. Embryol. 191:297-310). In dorsoventral patterning of Drosophila, expression of pox meso, an insect gene with high sequence similarity to Pax1, is repressed by decapentaplegic (dpp) in dorsal mesoderm and, thus, is restricted to a discrete ventral domain (Staehling-Hampton et al. [1994] Nature 372:783-786). In the chick wing, cells expressing a vertebrate homolog of dpp, bone morphogenetic protein 4 (Bmp4), abut the Pax1 domain, suggesting a similar relationship between homologous genes in both vertebrates and invertebrates. Here, we show that two BMPs (BMP4, and BMP2, also highly related to dpp) can repress Pax1 in the developing chick wing. Chick wing bud cells expressing Pax1 give rise to the shoulder girdle. Cells in an equivalent position in the mouse forelimb also express Pax1, and Pax1 mutant mice display shoulder girdle defects. Similarly in chick embryos, girdle defects are produced by treatments with signalling molecules that lead to expression of BMPs, which subsequently reduce Pax1 expression in the limb bud. Recently, BMP4 has been shown to inhibit Pax1 expression in the developing trunk (Monsoro-Burq et al. [1996] Development 122:3607-3616) and Pax9 expression in developing teeth (Neubüser et al. [1997] Cell 90:247-255). Thus, a property of BMPs appears to be to regulate pox meso homologs negatively and, thus, limit their expression domains.

FBP WW domains and the Abl SH3 domain bind to a specific class of proline-rich ligands.

WW domains are conserved protein motifs of 38-40 amino acids found in a broad spectrum of proteins. They mediate protein-protein interactions by binding proline-rich modules in ligands. A 10 amino acid proline-rich portion of the morphogenic protein, formin, is bound in vitro by both the WW domain of the formin-binding protein 11 (FBP11) and the SH3 domain of Abl. To explore whether the FBP11 WW domain and Abl SH3 domain bind to similar ligands, we screened a mouse limb bud expression library for putative ligands of the FBP11 WW domain. In so doing, we identified eight ligands (WBP3 through WBP10), each of which contains a proline-rich region or regions. Peptide sequence comparisons of the ligands revealed a conserved motif of 10 amino acids that acts as a modular sequence binding the FBP11 WW domain, but not the WW domain of the putative signal transducing factor, hYAP65. Interestingly, the consensus ligand for the FBP11 WW domain contains residues that are also required for binding by the Abl SH3 domain. These findings support the notion that the FBP11 WW domain and the Abl SH3 domain can compete for the same proline-rich ligands and suggest that at least two subclasses of WW domains exist, namely those that bind a PPLP motif, and those that bind a PPXY motif.

Formin binding proteins bear WWP/WW domains that bind proline-rich peptides and functionally resemble SH3 domains.

The formins, proteins involved in murine limb and kidney development, contain a proline-rich region that matches consensus sequences for Src homology 3 (SH3) ligands. To identify proteins that interact with formins, we used this proline-rich region to screen mouse limb bud expression libraries for formin binding proteins (FBPs). As expected, we found one class of FBPs that contains SH3 domains, including two novel members of this class. In addition, however, we also found a novel class of FBPs that contains one or two copies of a 26 amino acid homology region that has been recently termed the WWP or WW motif. We demonstrate that WWP/WW domains as short as 26 amino acids can act as modular protein-binding interfaces that bind with high affinity to proline-rich sequences that are similar and, in some cases, identical to SH3 ligands. Furthermore, we find that the WWP/WW domain can compete with the Abl SH3 domain in binding a proline-rich peptide present in formin. Our results suggest that these novel protein interaction domains can perform functions similar to those of SH3 domains and, thus, might regulate SH3 interactions with target proteins through competitive binding.

The Distal Human myoD Enhancer Sequences Direct Unique Muscle-Specific Patterns of lacZ Expression during Mouse Development

Transgenic mice carrying the bacterial lacZ reporter gene under the control of the regulatory elements of the human myoD gene have been produced. The developmental expression of the myoD reporter transgene in somites, limb buds, visceral arches, and cephalocervical regions was studied in transgenic embryos by β-gal staining. In somites, the spatiotemporal pattern of transgene expression was different from other muscle-specific regulatory and structural genes and revealed that myoD-expressing cells arise in distinct patterns in somites that are dependent on position along the anterior-posterior (AP) body axis (occipital and cervical vs thoracic and more posterior myotomes). Transgene expression did not follow a strict anterior to posterior sequence of activation and therefore was not strictly correlated with somite developmental age. Moreover, the pattern of transgene expression along the dorsal-ventral myotomal axis was dependent on somite position along the anterior-posterior axis. While myoD expression is first detected after the myotome is well-formed, transgene expression in the dorsal and ventral medial lips of the dermatome suggests a function for myoD in the expansion of the myotome. Whole-mount in situ hybridization confirmed that these unique patterns of transgene expression in somites, as well as expression in limb buds, visceral arches, and other myogenic centers, are concordant with the distribution of endogenous myoD transcripts. These results shed new light on the developmental differences between myotomes at different positions along the AP and DV axis and demonstrate a unique axial pattern of somitic myoD expression, suggesting a specific role of myoD in myotome lineage determination and differentiation.

Position-dependence of retinoic acid receptor-β gene expression in the chick limb bud

Retinoic acid and 3,4-didehydroretinoic acid are metabolites of vitamin A that can induce duplications and other malformations when locally applied to the anterior margin of the chick limb bud. There is evidence that they may be natural signaling substances in the limb bud. Both compounds are thought to act by binding to ligand-dependent transcription factors that belong to the steroid/thyroid hormone nuclear receptor superfamily. In situ hybridization analyses show that in the mesenchyme of the chick wing bud between embryonic stages 20 and 27, retinoic acid receptor-β (RAR-β) transcripts are restricted to the proximal region of the bud and are present at highest levels in the region of the limb bud mesenchyme that contributes to the shoulder. We have performed grafting experiments in order to examine whether RAR-β gene expression in limb bud mesenchyme cells is cell-autonomous or whether it is dependent upon the cell's position within the limb bud. When tissue from the proximal region of the stage 22 wing bud, which contains high levels of RAR-β transcripts, was grafted to the distal tip of the bud, RAR-β transcripts were undetectable in the graft 6 hr later. When tissue from the distal tip of the bud was grafted to a proximal site, most of the grafts exhibited a slight increase in the level of RAR-β transcripts, which was detectable 6 hr after grafting. However, the levels of RAR-β transcripts in these grafts never approached those found in the proximal core of the bud. These data indicate that RAR-β gene expression in the chick wing bud is position-dependent in that it is repressed at the distal tip of the bud and partially activated by grafting distal tissue to a proximal site. However, accumulation of RAR-β transcripts to high levels appears to be a characteristic of mesenchyme that was initially specified to form proximal structures.