Recent advances in hedgehog signalling.

Hedgehog (HH) proteins are an important class of secreted intercellular signals. The HH signal-transduction pathway is not fully understood, but a number of novel features have been elucidated recently. It is now clear that, during processing to generate an active signal, Drosophila HH proteins become covalently linked to cholesterol and are thereby largely tethered to the cell surface. HH signalling could therefore be affected by cholesterol metabolism. In addition, the pathway downstream of receptor binding involves a unique signalling complex containing the transcription factor CUBITUS INTERRUPTUS (CI), which becomes dissociated from microtubules in response to HH. This review discusses these new findings and their implications for HH signalling.

Role of Pitx1 upstream of Tbx4 in specification of hindlimb identity.

In spite of recent breakthroughs in understanding limb patterning, the genetic factors determining the differences between the forelimb and the hindlimb have not been understood. The genes Pitx1 and Tbx4 encode transcription factors that are expressed throughout the developing hindlimb but not forelimb buds. Misexpression of Pitx1 in the chick wing bud induced distal expression of Tbx4, as well as HoxC10 and HoxC11, which are normally restricted to hindlimb expression domains. Wing buds in which Pitx1 was misexpressed developed into limbs with some morphological characteristics of hindlimbs: the flexure was altered to that normally observed in legs, the digits were more toe-like in their relative size and shape, and the muscle pattern was transformed to that of a leg.

Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein.

Proper regulation of chondrocyte differentiation is necessary for the morphogenesis of skeletal elements, yet little is known about the molecular regulation of this process. A chicken homolog of Indian hedgehog (Ihh), a member of the conserved Hedgehog family of secreted proteins that is expressed during bone formation, has now been isolated. Ihh has biological properties similar to those of Sonic hedgehog (Shh), including the ability to regulate the conserved targets Patched (Ptc) and Gli. Ihh is expressed in the prehypertrophic chondrocytes of cartilage elements, where it regulates the rate of hypertrophic differentiation. Misexpression of Ihh prevents proliferating chondrocytes from initiating the hypertrophic differentiation process. The direct target of Ihh signaling is the perichondrium, where Gli and Ptc flank the expression domain of Ihh. Ihh induces the expression of a second signal, parathyroid hormone-related protein (PTHrP), in the periarticular perichondrium. Analysis of PTHrP (-/-) mutant mice indicated that the PTHrP protein signals to its receptor in the prehypertrophic chondrocytes, thereby blocking hypertrophic differentiation. In vitro application of Hedgehog or PTHrP protein to normal or PTHrP (-/-) limb explants demonstrated that PTHrP mediates the effects of Ihh through the formation of a negative feedback loop that modulates the rate of chondrocyte differentiation.

Distinct WNT pathways regulating AER formation and dorsoventral polarity in the chick limb bud.

The apical ectodermal ridge (AER) is an essential structure for vertebrate limb development. Wnt3a is expressed during the induction of the chick AER, and misexpression of Wnt3a induces ectopic expression of AER-specific genes in the limb ectoderm. The genes beta-catenin and Lef1 can mimic the effect of Wnt3a, and blocking the intrinsic Lef1 activity disrupts AER formation. Hence, Wnt3a functions in AER formation through the beta-catenin/LEF1 pathway. In contrast, neither beta-catenin nor Lef1 affects the Wnt7a-regulated dorsoventral polarity of the limb. Thus, two related Wnt genes elicit distinct responses in the same tissues by using different intracellular pathways.

Constructive antagonism in limb development.

Several advances have been made in our understanding of the control of the growth and patterning of embryonic limbs. Development of the vertebrate limb is dependent on reciprocal interactions between the ectoderm and mesoderm that regulate the structure and function of the apical ectodermal ridge. One key component of this regulatory program appears to be the precise control of signaling by members of the bone morphogenetic protein family via multiple antagonistic interactions.

The role of homeobox genes in limb development.

Intensive study of the development of the vertebrate limb has led to a conceptual framework for understanding the specification of a limb primordium, the outgrowth of those cells and their organization and differentiation into a functional appendage. During the past few years, a number of homeobox-containing genes have been identified that are likely to play controlling roles in each of these events.

Mechanisms of limb patterning.

The development of the vertebrate limb requires the coordinated action of multiple signals to achieve the proper arrangement of adult tissues. Recently, several molecules have been identified which play central roles in patterning of the limb bud. Sonic hedgehog, a homolog of the Drosophila segment polarity gene hedgehog, is likely to regulate anterior/posterior pattern formation. FGF-2 and FGF-4, members of the fibroblast growth factor family, have been shown to provide important signals for limb bud outgrowth and to indirectly regulate proximal/distal patterning. Some candidate effectors of the activity of Sonic hedgehog and of FGFs are known, including members of the clustered Hox genes.

Adaptation of a retrovirus as a eucaryotic vector transmitting the herpes simplex virus thymidine kinase gene.

We investigated the feasibility of using retroviruses as vectors for transferring DNA sequences into animal cells. The thymidine kinase (tk) gene of herpes simplex virus was chosen as a convenient model. The internal BamHI fragments of a DNA clone of Moloney leukemia virus (MLV) were replaced with a purified BamHI DNA segment containing the tk gene. Chimeric genomes were created carrying the tk insert in both orientations relative to the MLV sequence. Each was transfected into TK- cells along with MLV helper virus, and TK+ colonies were obtained by selection in the presence of hypoxanthine, aminopterin, and thymidine (HAT). Virus collected from TK+-transformed, MLV producer cells passed the TK+ phenotype to TK- cells. Nonproducer cells were isolated, and TK+ transducing virus was subsequently rescued from them. The chimeric virus showed single-hit kinetics in infections. Virion and cellular RNA and cellular DNA from infected cells were all shown to contain sequences which hybridized to both MLV- and tk-specific probes. The sizes of these sequences were consistent with those predicted for the chimeric virus. In all respects studied, the chimeric MLV-tk virus behaved like known replication-defective retroviruses. These experiments suggest great general applicability of retroviruses as eucaryotic vectors.

Antibodies to the ras gene product inhibit adenylate cyclase and accelerate progesterone-induced cell division in Xenopus laevis oocytes.

Microinjection of monoclonal antibodies (lines 238, 172, and 259) directed against the ras gene product, p21, into Xenopus laevis oocytes accelerated progesterone-induced germinal vesicle breakdown. Antibody 238 had the greatest effect on the acceleration of progesterone-induced oocyte maturation, and this effect was correlated with in vitro inhibition of adenylate cyclase (EC 4.6.1.1) activity in a concentration-dependent manner. Inhibition of adenylate cyclase by antibody 238 was also measured in membranes prepared from oocytes pretreated with either cholera toxin or pertussis toxin. These results suggest a role for the ras gene product in the regulation of vertebrate cell adenylate cyclase activity.

Chick Barx2b, a marker for myogenic cells also expressed in branchial arches and neural structures.

We have isolated a new chicken gene, cBarx2b, which is related to mBarx2 in sequence, although the expression patterns of the two genes are quite different from one another. The cBarx2b gene is expressed in craniofacial structures, regions of the neural tube, and muscle groups in the limb, neck and cloaca. Perturbation of anterior muscle pattern by application of Sonic Hedgehog protein results in a posteriorization of cBarx2b expression.

Similar expression and regulation of Gli2 and Gli3 in the chick limb bud.

Gli genes encode a family of zinc finger transcription factors that mediate signaling by Hedgehog proteins. We have cloned the chick Gli3 gene and studied its expression in developing chick limbs. Gli3 expression is highly similar to that of chick Gli2. Gli3 mRNA is evenly distributed in the early limb mesenchyme and subsequently downregulated in the posterior mesenchyme by the polarizing activity of Sonic hedgehog. At later stages, Gli3 is expressed in the distal limb mesenchyme.

Recapitulation of signals regulating embryonic bone formation during postnatal growth and in fracture repair.

A number of proteins have recently been identified which play roles in regulating bone development. One important example is Indian hedgehog (Ihh) which is secreted by the prehyprtrophic chondrocytes. Ihh acts as an activator of a second secreted factor, parathyroid hormone-related protein (PTHrP), which, in turn, negatively regulates the rate of chondrocyte differentiation. Here we examine the expression of these genes and their molecular targets during different stages of bone development. In addition to regulating PTHrP expression in the perichondrium, we find evidence that Ihh may also act on the chondrocytes themselves at particular stages. As bone growth continues postnatally in mammals and the developmental process is reactivated during fracture repair, understanding the molecular basis regulating bone development is of medical relevance. We find that the same molecules that regulate embryonic endochondral ossification are also expressed during postnatal bone growth and fracture healing, suggesting that these processes are controlled by similar mechanisms.

Comparative biological responses to human Sonic, Indian, and Desert hedgehog.

A comprehensive comparison of Sonic (Shh), Indian (Ihh), and Desert (Dhh) hedgehog biological activities has not previously been undertaken. To test whether the three higher vertebrate Hh proteins have distinct biological properties, we compared recombinant forms of the N-terminal domains of human Shh, Ihh, and Dhh in a variety of cell-based and tissue explant assays in which their activities could be assessed at a range of concentrations. While we observed that the proteins were similar in their affinities for the Hh-binding proteins; Patched (Ptc) and Hedgehog-interacting protein (Hip), and were equipotent in their ability to induce Islet-1 in chick neural plate explant; there were dramatic differences in their potencies in several other assays. Most dramatic were the Hh-dependent responses of C3H10T1/2 cells, where relative potencies ranged from 80nM for Shh, to 500nM for Ihh, to >5microM for Dhh. Similar trends in potency were seen in the ability of the three Hh proteins to induce differentiation of chondrocytes in embryonic mouse limbs, and to induce the expression of nodal in the lateral plate mesoderm of early chick embryos. However, in a chick embryo digit duplication assay used to measure polarizing activity, Ihh was the least active, and Dhh was almost as potent as Shh. These findings suggest that a mechanism for fine-tuning the biological actions of Shh, Ihh, and Dhh, exists beyond the simple temporal and spatial control of their expression domains within the developing and adult organism.

Goosecoid misexpression alters the morphology and Hox gene expression of the developing chick limb bud.

The homeobox-containing gene goosecoid (gsc) has been implicated in a variety of embryonic processes from gastrulation to rib patterning. We have analyzed the role it plays during chick limb development. Expression is initially observed at stage 20 in a proximal-anterior-ventral domain of the early limb bud which expands during subsequent stages. Later in limb development a second domain of expression appears distally which resolves to regions which surround the condensing cartilage. In order to understand the function of gsc in limb development, we have examined the effect of misexpressing gsc throughout the limb. Two striking phenotypes are observed. The first, evident at stage 24, is an alteration in the angle of femur outgrowth from the main body axis. The second, which can be detected at day 10 of development, is an overall decrease in the size of the limb with bones that are small, misshapen and bent. These phenotypes correlate with a decrease in levels of Hox gene expression in gsc-infected limb buds. From these results we suggest that gsc may normally function to regulate growth and patterning of the limb, perhaps through regulation of Hox gene expression.

Cloning and expression of a novel cysteine-rich secreted protein family member expressed in thyroid and pancreatic mesoderm within the chicken embryo.

We have isolated a new chicken gene that is a member of the cysteine-rich secreted protein family (CRISP). The CRISP family is composed of over 70 members that are found in many phyla of organisms, including: vertebrates, plants, fungi, yeast, and insects. Here we describe the cloning of a novel member of this family, SugarCrisp, and its expression pattern throughout chicken embryogenesis. We also describe its utility as a marker of thyroid and pancreatic mesoderm in the developing chicken embryo and its expression within the human and mouse in glandular tissue.

A cellular lineage analysis of the chick limb bud.

The chick limb bud has been used as a model system for studying pattern formation and tissue development for more than 50 years. However, the lineal relationships among the different cell types and the migrational boundaries of individual cells within the limb mesenchyme have not been explored. We have used a retroviral lineage analysis system to track the fate of single limb bud mesenchymal cells at different times in early limb development. We find that progenitor cells labeled at stage 19-22 can give rise to multiple cell types including clones containing cells of all five of the major lateral plate mesoderm-derived tissues (cartilage, perichondrium, tendon, muscle connective tissue, and dermis). There is a bias, however, such that clones are more likely to contain the cell types of spatially adjacent tissues such as cartilage/perichondrium and tendon/muscle connective tissue. It has been recently proposed that distinct proximodistal segments are established early in limb development; however our analysis suggests that there is not a strict barrier to cellular migration along the proximodistal axis in the early stage 19-22 limb buds. Finally, our data indicate the presence of a dorsal/ventral boundary established by stage 16 that is inhibitory to cellular mixing. This boundary is demarcated by the expression of the LIM-homeodomain factor lmx1b.

Isolation of potential vertebrate limb-identity genes.

Forelimbs and hindlimbs of tetrapods have different morphological patterns. One plausible explanation for the difference is that the cells that give rise to the limbs differentially express genes which control their pattern of development. Amphibian limb regeneration is an excellent system to test this hypothesis, since the same ultimate morphology is attained in regeneration as through embryogenesis. Using a combination of homeobox probes and differential screening, I have isolated two newt genes which are differentially expressed in regenerating forelimbs and hindlimbs. One of these genes displays properties expected of a gene involved in controlling limb morphology, including expression in mesodermal tissue and constancy of expression upon transplantation. Based on sequence analysis, this gene appears to be homologous to a homeobox-containing gene previously isolated from frog and human libraries.

Why we have (only) five fingers per hand: hox genes and the evolution of paired limbs.

Limb development has long been a model system for studying vertebrate pattern formation. The advent of molecular biology has allowed the identification of some of the key genes that regulate limb morphogenesis. One important class of such genes are the homeobox-containing, or Hox genes. Understanding of the roles these genes play in development additionally provides insights into the evolution of limb pattern. Hox gene expression patterns divide the embryonic limb bud into five sectors along the anterior/posterior axis. The expression of specific Hox genes in each domain specifies the developmental fate of that region. Because there are only five distinct Hox-encoded domains across the limb bud there is a developmental constraint prohibiting the evolution of more than five different types of digits. The expression patterns of Hox genes in modern embryonic limb buds also gives clues to the shape of the ancestral fin field from which the limb evolved, hence elucidating the evolution of the tetrapod limb.