Pleiotropic patterning response to activation of Shh signaling in the limb apical ectodermal ridge.

Sonic hedgehog (Shh) signaling in the limb plays a central role in coordination of limb patterning and outgrowth. Shh expression in the limb is limited to the cells of the zone of polarizing activity (ZPA), located in posterior limb bud mesoderm. Shh is not expressed by limb ectoderm or apical ectodermal ridge (AER), but recent studies suggest a role for AER-Shh signaling in limb patterning.

Direct and progressive differentiation of human embryonic stem cells into the chondrogenic lineage.

Treatment of common and debilitating degenerative cartilage diseases particularly osteoarthritis is a clinical challenge because of the limited capacity of the tissue for self-repair. Because of their unlimited capacity for self-renewal and ability to differentiate into multiple lineages, human embryonic stem cells (hESCs) are a potentially powerful tool for repair of cartilage defects. The primary objective of the present study was to develop culture systems and conditions that enable hESCs to directly and uniformly differentiate into the chondrogenic lineage without prior embryoid body (EB) formation, since the inherent cellular heterogeneity of EBs hinders obtaining homogeneous populations of chondrogenic cells that can be used for cartilage repair.

Conditional inactivation of Has2 reveals a crucial role for hyaluronan in skeletal growth, patterning, chondrocyte maturation and joint formation in the developing limb.

The glycosaminoglycan hyaluronan (HA) is a structural component of extracellular matrices and also interacts with cell surface receptors to directly influence cell behavior. To explore functions of HA in limb skeletal development, we conditionally inactivated the gene for HA synthase 2, Has2, in limb bud mesoderm using mice that harbor a floxed allele of Has2 and mice carrying a limb mesoderm-specific Prx1-Cre transgene. The skeletal elements of Has2-deficient limbs are severely shortened, indicating that HA is essential for normal longitudinal growth of all limb skeletal elements.

Studies on the role of Dlx5 in regulation of chondrocyte differentiation during endochondral ossification in the developing mouse limb.

The homeodomain transcription factor Dlx5 has been implicated in the regulation of chondrocyte and osteoblast differentiation during endochondral ossification in the developing limb. In a gain-of-function approach to directly investigate the role of Dlx5 in chondrocyte maturation, we have used cartilage-specific Col2a1-Dlx5 promoter/enhancer constructs to target overexpression of Dlx5 to the differentiating cartilage models of the limbs of transgenic mice. Targeted overexpression of Dlx5 in cartilage rudiments results in the formation of shortened skeletal elements containing excessive numbers of hypertrophic chondrocytes and expanded domains of expression of Ihh and type X collagen, molecular markers of hypertrophic maturation.

Hyaluronan in limb morphogenesis.

Hyaluronan (HA) is a large glycosaminoglycan that is not only a structural component of extracellular matrices, but also interacts with cell surface receptors to promote cell proliferation, migration, and intracellular signaling. HA is a major component of the extracellular matrix of the distal subapical mesenchymal cells of the developing limb bud that are undergoing proliferation, directed migration, and patterning in response to the apical ectodermal ridge (AER), and has the functional potential to be involved in these processes. Here we show that the HA synthase Has2 is abundantly expressed by the distal subridge mesodermal cells of the chick limb bud and also by the AER itself.

Expression of Dlx5 and Dlx6 during specification of the elbow joint.

The onset of elbow joint formation in the developing limb is characterized morphologically by the conversion of differentiated chondrocytes at the site of incipient joint formation into the densely packed flattened cells of the joint interzone. However, experimental studies have indicated that the elbow joint is specified well before joint interzone formation by a distinctive population of precursor cells located at the site in the developing limb bud at which the elbow joint will subsequently form. Here we show that during specification of the elbow joint in the chick limb bud, the homeodomain transcription factors Dlx5 and Dlx6 are highly expressed by a discrete group of cells that encompass the prospective elbow joint.

Heparan sulfate proteoglycans including syndecan-3 modulate BMP activity during limb cartilage differentiation.

Bone morphogenetic proteins (BMPs) are involved in multiple aspects of limb development including regulation of cartilage differentiation. Several BMPs bind strongly to heparin, and heparan sulfate proteoglycans (HSPGs) at the cell surface or in the extracellular matrix have recently been implicated as modulators of BMP signaling in some developing systems. Here we have explored the role of HSPGs in regulating BMP activity during limb chondrogenesis by evaluating the effects of exogenous heparan sulfate (HS), heparitinase treatment, and overexpression of the HSPG syndecan-3 on the ability of BMP2 to modulate the chondrogenic differentiation of limb mesenchymal cells in micromass culture.

Function of BMPs in the apical ectoderm of the developing mouse limb.

Several bone morphogenetic proteins (BMPs) are expressed in the apical ectodermal ridge (AER), a critical signaling center that directs the outgrowth and patterning of limb mesoderm, but little is known about their function. To study the functions of apical ectodermal BMPs, an AER-specific promoter element from the Msx2 gene was used to target expression of the potent BMP antagonist noggin to the apical ectoderm of the limbs of transgenic mice. Msx2-noggin mutant mice have severely malformed limbs characterized by syndactyly, postaxial polydactyly, and dorsal transformations of ventral structures indicated by absence of ventral footpads and presence of supernumerary ventral nails.

Dlx5 is a positive regulator of chondrocyte differentiation during endochondral ossification.

The process of endochondral ossification in which the bones of the limb are formed after generation of cartilage models is dependent on a precisely regulated program of chondrocyte maturation. Here, we show that the homeobox-containing gene Dlx5 is expressed at the onset of chondrocyte maturation during the conversion of immature proliferating chondrocytes into postmitotic hypertrophying chondrocytes, a critical step in the maturation process. Moreover, retroviral misexpression of Dlx5 during differentiation of the skeletal elements of the chick limb in vivo results in the formation of severely shortened skeletal elements that contain excessive numbers of hypertrophying chondrocytes which extend into ectopic regions, including sites normally occupied by immature chondrocytes.

Distribution and possible function of an adrenomedullin-like peptide in the developing chick limb bud.

Adrenomedullin (AM) is a multifunctional peptide that exhibits discrete domains of expression during mouse embryogenesis consistent with a role in regulating growth and differentiation during morphogenesis. Here we report that AM immunoreactivity is present at high levels throughout the apical ectodermal ridge (AER) of the chick limb bud as the AER is directing the outgrowth and patterning of underlying limb mesoderm. Immunostaining is particularly strong along the surfaces of the contiguous cells of the AER.

Overexpression of Dlx5 in chicken calvarial cells accelerates osteoblastic differentiation.

Our laboratory and others have shown that a homeodomain protein binding site plays an important role in transcription of the Collal gene in osteoblasts. This suggests that homeodomain proteins have an important role in osteoblast differentiation. We have investigated the role of Dlx5 in osteoblastic differentiation.

Studies on the role of Cux1 in regulation of the onset of joint formation in the developing limb.

Joint formation, the onset of which is characterized by the segmentation of continuous skeletal rudiments into two or more separate elements, is a fundamental aspect of limb pattern formation, playing a critical role in determining the size, shape, and number of individual skeletal elements. Joint formation is initiated by conversion of differentiated chondrocytes at sites of presumptive joints into densely packed nonchondrogenic cells of the joint interzone. This conversion is accompanied by loss of Alcian blue-staining cartilage matrix and downregulation of cartilage-specific gene expression.