Pattern formation in epithelial development: the vertebrate limb and feather bud spacing

The ectoderm of the vertebrate limb and feather bud are epithelia that provide good models for epithelial patterning in vertebrate development. At the tip of chick and mouse limb buds is a thickening, the apical ectodermal ridge, which is essential for limb bud outgrowth. The signal from the ridge to the underlying mesoderm involves fibroblast growth factors. The non-ridge ectoderm specifies the dorsoventral pattern of the bud and Wnt7a is a dorsalizing signal. The development of the ridge involves an interaction between dorsal cells that express radical fringe and those that do not. There are striking similarities between the signals and genes involved in patterning the limb ectoderm and the epithelia of the Drosophila imaginal disc that gives rise to the wing. The spacing of feather buds involves signals from the epidermis to the underlying mesenchyme, which again include Wnt7a and fibroblast growth factors.     

The limb field mesoderm determines initial limb bud anteroposterior asymmetry and budding independent of sonic hedgehog or apical ectodermal gene expressions

We have analyzed the pattern of expression of several genes implicated in limb initiation and outgrowth using limbless chicken embryos. We demonstrate that the expressions of the apical ridge associated genes, Fgf-8, Fgf-4, Bmp-2 and Bmp-4, are undetectable in limbless limb bud ectoderm; however, FGF2 protein is present in the limb bud ectoderm. Shh expression is undetectable in limbless limb bud mesoderm. Nevertheless, limbless limb bud mesoderm shows polarization manifested by the asymmetric expression of Hoxd-11, -12 and -13, Wnt-5a and Bmp-4 genes. The posterior limbless limb bud mesoderm, although not actually expressing Shh, is competent to express it if supplied with exogenous FGF or transplanted to a normal apical ridge environment, providing further evidence of mesodermal asymmetry. Exogenous FGF applied to limbless limb buds permits further growth and determination of recognizable skeletal elements, without the development of an apical ridge. However, the cells competent to express Shh do so at reduced levels; nevertheless, Bmp-2 is then rapidly expressed in the posterior limbless mesoderm. limbless limb buds appear as bi-dorsal structures, as the entire limb bud ectoderm expresses Wnt-7a, a marker for dorsal limb bud ectoderm; the ectoderm fails to express En-1, a marker of ventral ectoderm. As expected, C-Lmx1, which is downstream of Wnt-7a, is expressed in the entire limbless limb bud mesoderm. We conclude that anteroposterior polarity is established in the initial limb bud prior to Shh expression, apical ridge gene expression or dorsal-ventral asymmetry. We propose that the initial pattern of gene expressions in the emergent limb bud is established by axial influences on the limb field. These permit the bud to emerge with asymmetric gene expression before Shh and the apical ridge appear. We report that expression of Fgf-8 by the limb ectoderm is not required for the initiation of the limb bud. The gene expressions in the pre-ridge limb bud mesoderm, as in the limb bud itself, are unstable without stimulation from the apical ridge and the polarizing region (Shh) after budding is initiated. We propose that the defect in limbless limb buds is the lack of a dorsal-ventral interface in the limb bud ectoderm where the apical ridge induction signal would be received and an apical ridge formed. These observations provide evidence for the hypothesis that the dorsal-ventral ectoderm interface is a precondition for apical ridge formation.     

The role of Alx-4 in the establishment of anteroposterior polarity during vertebrate limb development

We have determined that Strong's Luxoid (lstJ) [corrected] mice have a 16 bp deletion in the homeobox region of the Alx-4 gene. This deletion, which leads to a frame shift and a truncation of the Alx-4 protein, could cause the polydactyly phenotype observed in lstJ [corrected] mice. We have cloned the chick homologue of Alx-4 and investigated its expression during limb outgrowth. Chick Alx-4 displays an expression pattern complementary to that of shh, a mediator of polarizing activity in the limb bud. Local application of Sonic hedgehog (Shh) and Fibroblast Growth Factor (FGF), in addition to ectodermal apical ridge removal experiments suggest the existence of a negative feedback loop between Alx-4 and Shh during limb outgrowth. Analysis of polydactylous mutants indicate that the interaction between Alx-4 and Shh is independent of Gli3, a negative regulator of Shh in the limb. Our data suggest the existence of a negative feedback loop between Alx-4 and Shh during vertebrate limb outgrowth.     

Analysis of the genetic pathway leading to formation of ectopic apical ectodermal ridges in mouse Engrailed-1 mutant limbs

The apical ectodermal ridge (AER), a rim of thickened ectodermal cells at the interface between the dorsal and ventral domains of the limb bud, is required for limb outgrowth and patterning. We have previously shown that the limbs of En1 mutant mice display dorsal-ventral and proximal-distal abnormalities, the latter being reflected in the appearance of a broadened AER and formation of ectopic ventral digits. A detailed genetic analysis of wild-type, En1 and Wnt7a mutant limb buds during AER development has delineated a role for En1 in normal AER formation. Our studies support previous suggestions that AER maturation involves the compression of an early broad ventral domain of limb ectoderm into a narrow rim at the tip and further show that En1 plays a critical role in the compaction phase. Loss of En1 leads to a delay in the distal shift and stratification of cells in the ventral half of the AER. At later stages, this often leads to development of a secondary ventral AER, which can promote formation of an ectopic digit. The second AER forms at the juxtaposition of the ventral border of the broadened mutant AER and the distal border of an ectopic Lmx1b expression domain. Analysis of En1/Wnt7a double mutants demonstrates that the dorsalizing gene Wnt7a is required for the formation of the ectopic AERs in En1 mutants and for ectopic expression of Lmx1b in the ventral mesenchyme. We suggest a model whereby, in En1 mutants, ectopic ventral Wnt7a and/or Lmx1b expression leads to the transformation of ventral cells in the broadened AER to a more dorsal phenotype. This leads to induction of a second zone of compaction ventrally, which in some cases goes on to form an autonomous secondary AER.     

Prevalence and correlates of symptomatic peripheral atherosclerosis in individuals with coronary heart disease and cholesterol levels less than 240 mg/dL: baseline results from the Cholesterol and Recurrent Events (CARE) Study

The formation of the digits in amniote vertebrates is accompanied by a massive degeneration process that accounts for the disappearance of the interdigital mesenchyme. The establishment of these areas of interdigital cell death (INZs) is concomitant with the flattening of the apical ectodermal ridge (AER), but a possible causal relationship between these processes has not been demonstrated. Recent studies have shown that the function of the AER can be substituted for by implantation of beads bearing either FGF-2 or FGF-4 into the apical mesoderm of the early limb bud. According to these observations, if the onset of INZs is triggered by the cessation of the AER function, local administration of FGFs to the interdigital tissue prior to cell death should delay or inhibit interdigit degeneration. In the present study we have confirmed this prediction. Implanting Affi-gel blue or heparin beads pre-absorbed with either FGF-2 or FGF-4 into the interdigital tissue of the chick leg bud in the stages prior to cell death stimulates cell proliferation and causes the formation of webbed digits. Vital staining with neutral red confirmed an intense temporal inhibition of interdigital cell death after FGF treatment. This inhibition of interdigital cell death was not accompanied by modifications in the pattern of expression of Msx-1 or Msx-2 genes, which in normal development display a domain of expression in the interdigital tissue preceding the onset of degeneration.     

Identification of a neuronal calmodulin-binding peptide, CAP-19, containing an IQ motif

During early stages of chick limb development, the homeobox-containing gene Msx-2 is expressed in the mesoderm at the anterior margin of the limb bud and in a discrete group of mesodermal cells at the midproximal posterior margin. These domains of Msx-2 expression roughly demarcate the anterior and posterior boundaries of the progress zone, the highly proliferating posterior mesodermal cells underneath the apical ectodermal ridge (AER) that give rise to the skeletal elements of the limb and associated structures. Later in development as the AER loses its activity, Msx-2 expression expands into the distal mesoderm and subsequently into the interdigital mesenchyme which demarcates the developing digits. The domains of Msx-2 expression exhibit considerably less proliferation than the cells of the progress zone and also encompass several regions of programmed cell death including the anterior and posterior necrotic zones and interdigital mesenchyme. We have thus suggested that Msx-2 may be in a regulatory network that delimits the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed. In the present study we show that ectopic expression of Msx-2 via a retroviral expression vector in the posterior mesoderm of the progress zone from the time of initial formation of the limb bud severely impairs limb morphogenesis. Msx-2-infected limbs are typically very narrow along the anteroposterior axis, are occasionally truncated, and exhibit alterations in the pattern of formation of skeletal elements, indicating that as a consequence of ectopic Msx-2 expression the morphogenesis of large portions of the posterior mesoderm has been suppressed. We further show that Msx-2 impairs limb morphogenesis by reducing cell proliferation and promoting apoptosis in the regions of the posterior mesoderm in which it is ectopically expressed. The domains of ectopic Msx-2 expression in the posterior mesoderm also exhibit ectopic expression of BMP-4, a secreted signaling molecule that is coexpressed with Msx-2 during normal limb development in the anterior limb mesoderm, the posterior necrotic zone, and interdigital mesenchyme. This indicates that Msx-2 regulates BMP-4 expression and that the suppressive effects of Msx-2 on limb morphogenesis might be mediated in part by BMP-4. These studies indicate that during normal limb development Msx-2 is a key component of a regulatory network that delimits the boundaries of the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed, thus restricting the outgrowth and formation of skeletal elements and associated structures to the progress zone. We also report that rather large numbers of apoptotic cells as well as proliferating cells are present throughout the AER during all stages of normal limb development we have examined, indicating that many of the cells of the AER are continuously undergoing programmed cell death at the same time that new AER cells are being generated by cell proliferation. Thus, a balance between cell proliferation and programmed cell death may play a very important role in maintaining the activity of the AER.     

Radical fringe positions the apical ectodermal ridge at the dorsoventral boundary of the vertebrate limb

Vertebrate limb outgrowth requires a structure called the apical ectodermal ridge, formation of which follows the previous establishment of the dorsoventral limb axis. Radical fringe is expressed in the dorsal ectoderm before the ridge appears, and is repressed by Engrailed-1, which is expressed in the ventral ectoderm. Misexpression of these genes indicates that a ridge is formed wherever there is a boundary between cells expressing and not expressing Radical fringe. Thus, as in Drosophila, Radical fringe positions the ridge at the dorsoventral limb boundary.     

Retinoic acid signaling is required during early chick limb development

In the chick limb bud, the zone of polarizing activity controls limb patterning along the anteroposterior and proximodistal axes. Since retinoic acid can induce ectopic polarizing activity, we examined whether this molecule plays a role in the establishment of the endogenous zone of polarizing activity. Grafts of wing bud mesenchyme treated with physiologic doses of retinoic acid had weak polarizing activity but inclusion of a retinoic acid-exposed apical ectodermal ridge or of prospective wing bud ectoderm evoked strong polarizing activity. Likewise, polarizing activity of prospective wing mesenchyme was markedly enhanced by co-grafting either a retinoic acid-exposed apical ectodermal ridge or ectoderm from the wing region. This equivalence of ectoderm-mesenchyme interactions required for the establishment of polarizing activity in retinoic acid-treated wing buds and in prospective wing tissue, suggests a role of retinoic acid in the establishment of the zone of polarizing activity. We found that prospective wing bud tissue is a high-point of retinoic acid synthesis. Furthermore, retinoid receptor-specific antagonists blocked limb morphogenesis and down-regulated a polarizing signal, sonic hedgehog. Limb agenesis was reversed when antagonist-exposed wing buds were treated with retinoic acid. Our results demonstrate a role of retinoic acid in the establishment of the endogenous zone of polarizing activity.     

Slug, a zinc finger gene previously implicated in the early patterning of the mesoderm and the neural crest, is also involved in chick limb development

The great advances made over the last few years in the identification of signalling molecules that pattern the limb bud along the three axes make the limb an excellent model system with which to study developmental mechanisms in vertebrates. The understanding of the signalling networks and their mutual interactions during limb development requires the characterisation of the corresponding downstream genes. In this study we report the expression pattern of Slug, a zinc-finger-containing gene of the snail family, during the development of the limb, and its regulation by distinct axial signalling systems. Slug expression is highly dynamic, and at different stages of limb development can be correlated with the zone of polarizing activity, the progress zone and the interdigital areas. We show that the maintenance of its expression is dependent on signals from the apical ectodermal ridge and independent of Sonic Hedgehog. We also report that, in the interdigit, apoptotic cells lie outside of the domains of Slug expression. The correlation of Slug expression with areas of undifferentiated mesenchyme at stages of tissue differentiation is consistent with its role in early development, in maintaining the mesenchymal phenotype and repressing differentiation processes. We suggest that Slug is involved in the epithelial-mesenchymal interactions that lead to the maintenance of the progress zone.     

Early chick limb cartilaginous elements possess polarizing activity and express hedgehog-related morphogenetic factors

Skeletal patterning and morphogenesis in the developing limb are thought to be regulated by instructive factors and cues from the zone of polarizing activity (ZPA), the apical ectodermal ridge (AER), and the dorsal ectoderm. However, the activities of the ZPA and AER dwindle early in embryogenesis and soon after ceases, when in fact the proximal skeletal elements are still rudimentary in structure and the more distal ones are yet to become recognizable. Thus, we asked whether the chondrocytes emerging within each mesenchymal condensation may themselves start expressing properties similar to those of ZPA and/or AER and, in so doing, may bring skeletal development to completion. Indeed, we found that the cartilaginous, but not precartilaginous, tissues in early chick limbs possess ZPA-like properties. They expressed an endogenous factor related to Sonic hedgehog (Shh), most likely Indian hedgehog (Ihh), and when fragments were grafted to the anterior margin of host stage 16-20 chick wing buds, they induced supernumerary skeletal elements (polarizing activity). The acquisition of polarizing activity by the cartilaginous structures followed clear proximo-to-distal and posterior-to-anterior routes. Thus, (1) stage 25 cartilaginous humerus had polarizing activity while stage 25 prospective radius did not, (2) posteriorly-located stage 29 ulna had stronger activity than anteriorly-located stage 29 radius, and (3) ulna's diaphysis had stronger activity at stage 29 than 31 while radius's diaphysis was stronger at stage 31 than 29. Prior to inducing extra digit formation, the cartilaginous grafts induced Hoxd-12 and Hoxd-13 gene expression in adjacent competent mesenchymal tissue. Strikingly, the cartilaginous grafts activity also expression of Shh and polarizing activity in adjacent mesenchyme, which ZPA grafts cannot do; thus, the cartilaginous structures displayed activities "upstream" of those of the ZPA. The results support our hypothesis that chondrocytes may themselves direct skeletal morphogenesis. In so doing and as a result of their inductive activities, the cells may also have an important role in the completion of limb patterning and morphogenesis.     

Relationship between dose, distance and time in Sonic Hedgehog-mediated regulation of anteroposterior polarity in the chick limb

Anteroposterior polarity in the vertebrate limb is thought to be regulated in response to signals derived from a specialized region of distal posterior mesenchyme, the zone of polarizing activity. Sonic Hedgehog (Shh) is expressed in the zone of polarizing activity and appears to mediate the action of the zone of polarizing activity. Here we have manipulated Shh signal in the limb to assess whether it acts as a long-range signal to directly pattern all the digits. Firstly, we demonstrate that alterations in digit development are dependent upon the dose of Shh applied. DiI-labeling experiments indicate that cells giving rise to the extra digits lie within a 300 microm radius of a Shh bead and that the most posterior digits come from cells that lie very close to the bead. A response to Shh involves a 12-16 hour period in which no irreversible changes in digit pattern occur. Increasing the time of exposure to Shh leads to specification of additional digits, firstly digit 2, then 3, then 4. Cell marking experiments demonstrate that cells giving rise to posterior digits are first specified as anterior digits and later adopt a more posterior character. To monitor the direct range of Shh signalling, we developed sensitive assays for localizing Shh by attaching alkaline phosphatase to Shh and introducing cells expressing these forms into the limb bud. These experiments demonstrate that long-range diffusion across the anteroposterior axis of the limb is possible. However, despite a dramatic difference in their diffusibility in the limb mesenchyme, the two forms of alkaline phosphatase-tagged Shh proteins share similar polarizing activity. Moreover, Shh-N (aminoterminal peptide of Shh)-coated beads and Shh-expressing cells also exhibit similar patterning activity despite a significant difference in the diffusibility of Shh from these two sources. Finally, we demonstrate that when Shh-N is attached to an integral membrane protein, cells transfected with this anchored signal also induce mirror-image pattern duplications in a dose-dependent fashion similar to the zone of polarizing activity itself. These data suggest that it is unlikely that Shh itself signals digit formation at a distance. Beads soaked in Shh-N do not induce Shh in anterior limb mesenchyme ruling out direct propagation of a Shh signal. However, Shh induces dose-dependent expression of Bmp genes in anterior mesenchyme at the start of the promotion phase. Taken together, these results argue that the dose-dependent effects of Shh in the regulation of anteroposterior pattern in the limb may be mediated by some other signal(s). BMPs are plausible candidates.     

A study of patients with Nelson''s syndrome

Tooth development is regulated by a reciprocal series of epithelial-mesenchymal interactions. Bmp4 has been identified as a candidate signalling molecule in these interactions, initially as an epithelial signal and then later at the bud stage as a mesenchymal signal (Vainio et al. [1993] Cell 75:45-58). A target gene for Bmp4 signalling is the homeobox gene Msx-1, identified by the ability of recombinant Bmp4 protein to induce expression in mesenchyme. There is, however, no evidence that Bmp4 is the endogenous inducer of Msx-1 expression. Msx-1 and Bmp-4 show dynamic, interactive patterns of expression in oral epithelium and ectomesenchyme during the early stages of tooth development. In this study, we compare the temporal and spatial expression of these two genes to determine whether the changing expression patterns of these genes are consistent with interactions between the two molecules. We show that changes in Bmp-4 expression precede changes in Msx-1 expression. At embryonic day (E)10.5-E11.0, expression patterns are consistent with BMP4 from the epithelium, inducing or maintaining Msx-1 in underlying mesenchyme. At E11.5, Bmp-4 expression shifts from epithelium to mesenchyme and is rapidly followed by localised up-regulation of Msx-1 expression at the sites of Bmp-4 expression. Using cultured explants of developing mandibles, we confirm that exogenous BMP4 is capable of replacing the endogenous source in epithelium and inducing Msx-1 gene expression in mesenchyme. By using noggin, a BMP inhibitor, we show that endogenous Msx-1 expression can be inhibited at E10.5 and E11.5, providing the first evidence that endogenous Bmp-4 from the epithelium is responsible for regulating the early spatial expression of Msx-1. We also show that the mesenchymal shift in Bmp-4 is responsible for up-regulating Msx-1 specifically at the sites of future tooth formation. Thus, we establish that a reciprocal series of interactions act to restrict expression of both genes to future sites of tooth formation, creating a positive feedback loop that maintains expression of both genes in tooth mesenchymal cells.