Andrea Vortkamp

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.

PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth

The PTH/PTHrP receptor binds to two ligands with distinct functions: the calcium-regulating hormone, parathyroid hormone (PTH), and the paracrine factor, PTH-related protein (PTHrP). Each ligand, in turn, is likely to activate more than one receptor. The functions of the PTH/PTHrP receptor were investigated by deletion of the murine gene by homologous recombination. Most PTH/PTHrP receptor (−/−) mutant mice died in mid-gestation, a phenotype not observed in PTHrP (−/−) mice, perhaps because of the effects of maternal PTHrP. Mice that survived exhibited accelerated differentiation of chondrocytes in bone, and their bones, grown in explant culture, were resistant to the effects of PTHrP and Sonic hedgehog. These results suggest that the PTH/PTHrP receptor mediates the effects of Indian Hedgehog and PTHrP on chondrocyte differentiation.

Interaction of FGF, Ihh/Pthlh, and BMP Signaling Integrates Chondrocyte Proliferation and Hypertrophic Differentiation

Mutations in fibroblast growth factor (FGF) receptor 3 lead to the human dwarfism syndrome achondroplasia. Using a limb culture system, we have analyzed the role of FGF signaling and its interaction with the Ihh/Pthlh and BMP pathways in regulating chondrocyte differentiation. In contrast to previous suggestions, we demonstrate that FGF signaling accelerates both the onset and the pace of hypertrophic differentiation. We furthermore found that FGF and BMP signaling act in an antagonistic relationship regulating chondrocyte proliferation, Ihh expression, and the process of hypertrophic differentiation. Importantly, BMP signaling rescues the reduced domains of proliferating and hypertrophic chondrocytes in a mouse model for achondroplasia. We propose a model in which the balance of BMP and FGF signaling adjusts the pace of the differentiation process to the proliferation rate.

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.

Gli3 acts as a repressor downstream of Ihh in regulating two distinct steps of chondrocyte differentiation

During endochondral ossification, the secreted growth factor Indian hedgehog (Ihh) regulates several differentiation steps. It interacts with a second secreted factor, parathyroid hormone-related protein (PTHrP), to regulate the onset of hypertrophic differentiation, and it regulates chondrocyte proliferation and ossification of the perichondrium independently of PTHrP. To investigate how the Ihh signal is translated in the different target tissues, we analyzed the role of the zinc-finger transcription factor Gli3, which acts downstream of hedgehog signals in other organs. Loss of Gli3 in Ihh mutants restores chondrocyte proliferation and delays the accelerated onset of hypertrophic differentiation observed in Ihh–/– mutants. Furthermore the expression of the Ihh target genes patched (Ptch) and PTHrP is reactivated in Ihh–/–;Gli3–/– mutants. Gli3 seems thus to act as a strong repressor of Ihh signals in regulating chondrocyte differentiation. In addition, loss of Gli3 in mice that overexpress Ihh in chondrocytes accelerates the onset of hypertrophic differentiation by reducing the domain and possibly the level of PTHrP expression. Careful analysis of chondrocyte differentiation in Gli3–/– mutants revealed that Gli3 negatively regulates the differentiation of distal, low proliferating chondrocytes into columnar, high proliferating cells. Our results suggest a model in which the Ihh/Gli3 system regulates two distinct steps of chondrocyte differentiation: (1) the switch from distal into columnar chondrocytes is repressed by Gli3 in a PTHrP-independent mechanism; (2) the transition from proliferating into hypertrophic chondrocytes is regulated by Gli3-dependent expression of PTHrP. Furthermore, by regulating distal chondrocyte differentiation, Gli3 seems to position the domain of PTHrP expression.

Essential Role for ADAM19 in Cardiovascular Morphogenesis

ABSTRACT Congenital heart disease is the most common form of human birth defects, yet much remains to be learned about its underlying causes. Here we report that mice lacking functional ADAM19 (mnemonic for a disintegrin and metalloprotease 19) exhibit severe defects in cardiac morphogenesis, including a ventricular septal defect (VSD), abnormal formation of the aortic and pulmonic valves, leading to valvular stenosis, and abnormalities of the cardiac vasculature. During mouse development, ADAM19 is highly expressed in the conotruncus and the endocardial cushion, structures that give rise to the affected heart valves and the membranous ventricular septum. ADAM19 is also highly expressed in osteoblast-like cells in the bone, yet it does not appear to be essential for bone growth and skeletal development. Most adam19−/− animals die perinatally, likely as a result of their cardiac defects. These findings raise the possibility that mutations in ADAM19 may contribute to human congenital heart valve and septal defects.

Sex-specific expression of an evolutionarily conserved male regulatory gene, DMRT1, in birds

Based on its Z-sex-chromosomal location and its structural homology to male sexual regulatory factors in humans (DMRT1 and DMRT2), Drosophila (Dsx), and Caenorhabditis elegans (Mab-3), chicken DMRT1 is an excellent candidate for a testis-determining factor in birds. The data we present provide further strong support for this hypothesis. By whole mount in situ hybridization chicken DMRT1 is expressed at higher levels in the male than in the female genital ridges during early stages of embryogenesis. Its expression becomes testis-specific after onset of sexual differentiation. Northern blot and RT PCR analysis showed that in adult birds DMRT1 is expressed exclusively in the testis. We propose that two gene dosages are required for testis formation in ZZ males, whereas expression from a single Z chromosome in ZW females leads to female sexual differentiation.

Deletion of GLI3 supports the homology of the human Greig cephalopolysyndactyly syndrome (GCPS) and the mouse mutant extra toes (Xt).

The dominant mouse mutant extra toes (Xt) is characterized by preaxial and postaxial polydactyly of the feet and a white belly spot. An interfrontal bone is present in the skull in 90% of heterozygotes compared with 50% of normal mice. Homozygous XtlXt embryos exhibit multiple skeletal defects, extreme polydactyly in both foreand hindlimbs, and malformations of the brain and the eye (Johnson 1967; Franz and Besecke 1991). Depending on the genetic background, Xt homozygotes die prenatally or perinatally (J ohnson 1967). An allelic but recessive syndrome is the anterior digit pattern deformity mutation (add), which is the result of a transgene integration (Pohl et al. 1990). In this case the malformations of the mutants are restricted to the forelimbs. Using DNA probes spanning the add transgene integration site, Po hI and coworkers (1990) could show that at least 80 kbp of surrounding DNA are deleted in Xt mice. On the basis of the similarity of the phenotype, Xt is considered the mouse homolog of the human autosomal dominant Greig cephalopolysyndactyly syndrome, GCPS (Winter and Huson 1988). The latter is characterized by polysyndactyly of hands and feet and mild craniofacial abnormalities (Gollop and Fontes 1985)_ The gene locus has been pinpointed to human Chromosome (Chr) 7p13 by different translocations and deletions associated with the disorder (Tommerup and Nielsen 1983; Kriiger et al. 1989; Wagner et al. 1990; Pettigrew et al. 1991; Vortkamp et al. 1991b). Using six hybrids from three GCPS translocation patients, we recently have demonstrated that the zinc finger gene GLI3 (Ruppert et al. 1988, 1990) is dis-

A mouse model of osteochondromagenesis from clonal inactivation of Ext1 in chondrocytes

We report a mouse model of multiple osteochondromas (MO), an autosomal dominant disease in humans, also known as multiple hereditary exostoses (MHE or HME) and characterized by the formation of cartilage-capped osseous growths projecting from the metaphyses of endochondral bones. The pathogenesis of these osteochondromas has remained unclear. Mice heterozygous for Ext1 or Ext2, modeling the human genotypes that cause MO, occasionally develop solitary osteochondroma-like structures on ribs [Lin et al. (2000) Dev Biol 224(2):299–311; Stickens et al. (2005) Development 132(22):5055–5068]. Rather than model the germ-line genotype, we modeled the chimeric tissue genotype of somatic loss of heterozygosity (LOH), by conditionally inactivating Ext1 via head-to-head loxP sites and temporally controlled Cre-recombinase in chondrocytes. These mice faithfully recapitulate the human phenotype of multiple metaphyseal osteochondromas. We also confirm homozygous disruption of Ext1 in osteochondroma chondrocytes and their origin in proliferating physeal chondrocytes. These results explain prior modeling failures with the necessity for somatic LOH in a developmentally regulated cell type.

Transcriptional networks controlling chondrocyte proliferation and differentiation during endochondral ossification.

During endochondral ossification bones are formed as cartilage templates in which chondrocytes proliferate, differentiate into hypertrophic chondrocytes and are gradually replaced by bone. Postnatally, remnants of embryonic chondrocytes remain in a restricted domain between the ossified regions of the bones forming the growth plate. The coordinated proliferation and differentiation of chondrocytes ensures the continuous elongation of the epiphyseal growth plates. The sequential changes between proliferation and differentiation are tightly regulated by secreted growth factors, which activate chondrocyte-specific transcription factors. Transcription factors that play critical roles in regulating cell-type-specific gene expression include Sox9, Gli2/3, and Runx2. The interaction of these transcription factors with general transcriptional regulators like histone-modifying enzymes provides an additional level of regulation to fine-tune the expression of target genes in different chondrocyte populations. This review will outline recent advances in the analysis of the complex transcriptional network that regulates the distinct steps of chondrocyte differentiation.