Robert A. Kosher

Wnt-5a and Wnt-7a are expressed in the developing chick limb bud in a manner suggesting roles in pattern formation along the proximodistal and dorsoventral axes

The Wnt gene family encodes a group of secreted signalling molecules that have been implicated in the regulation of cell fate and pattern formation during embryogenesis. We have examined the patterns of expression of two members of the chicken Wnt family, Wnt-5a and Wnt-7a, during development of the chick limb bud. Wnt-5a is expressed in the apical ectodermal ridge which directs outgrowth of limb mesoderm. Wnt-5a also exhibits three quantitatively distinct domains of expression along the proximodistal (PD) axis of the limb mesoderm that may correspond to the regions which will give rise to the three distinct PD segments of the limb, the autopod, zeugopod, and stylopod. In contrast, Wnt-7a expression in the limb bud is specifically limited to the dorsal ectoderm. These observations suggest possible roles for Wnt-5a and Wnt-7a in pattern formation along the PD and dorsoventral axes of the developing chick limb bud. In addition, Wnt-5a and Wnt-7a exhibit spatially discrete domains of expression in several other regions of the chick embryo consistent with developmental roles for these genes in a variety of other tissues.

Promotion of embryonic chick limb cartilage differentiation by transforming growth factor-β☆

This study represents a first step in investigating the possible involvement of transforming growth factor-beta (TGF-beta) in the regulation of embryonic chick limb cartilage differentiation. TGF-beta 1 and 2 (1-10 ng/ml) elicit a striking increase in the accumulation of Alcian blue, pH 1-positive cartilage matrix, and a corresponding twofold to threefold increase in the accumulation of 35S-sulfate- or 3H-glucosamine-labeled sulfated glycosaminoglycans (GAG) by high density micromass cultures prepared from the cells of whole stage 23/24 limb buds or the homogeneous population of chondrogenic precursor cells comprising the distal subridge mesenchyme of stage 25 wing buds. Moreover, TGF-beta causes a striking (threefold to sixfold) increase in the steady-state cytoplasmic levels of mRNAs for cartilage-characteristic type II collagen and the core protein of cartilage-specific proteoglycan. Only a brief (2 hr) exposure to TGF-beta at the initiation of culture is sufficient to stimulate chondrogenesis, indicating that the growth factor is acting at an early step in the process. Furthermore, TGF-beta promotes the formation of cartilage matrix and cartilage-specific gene expression in low density subconfluent spot cultures of limb mesenchymal cells, which are situations in which little, or no chondrogenic differentiation normally occurs. These results provide strong incentive for considering and further investigating the role of TGF-beta in the control of limb cartilage differentiation.

Expression of the chicken homeobox-containing gene GHox-8 during embryonic chick limb development

Homeobox-containing genes are thought to be involved in the regulation of pattern formation and specification of positional information during vertebrate limb development. Because of its accessibility to microsurgical manipulation, the developing chick limb bud provides a powerful system for investigating the role of homeobox-containing genes in patterning events. We report the isolation from a chick limb bud cDNA library of a chicken homeobox-containing cDNA, which on the basis of its nucleotide and deduced amino acid sequences has been identified as the chicken cognate of mouse Hox-8. The gene encoding this chicken (Gallus) homeobox-containing cDNA has been designated GHox-8, and is a member of a family of vertebrate homeobox-containing genes that are highly similar in sequence to the Drosophila msh gene. GHox-8 encodes an mRNA transcript of about 3 kb that is expressed at several early stages of chick limb development. In situ and dot-blot hybridization analyses have revealed that GHox-8 is expressed in limb bud mesoderm in a temporal and spatial fashion consistent with its involvement in specifying anterior positional identity. At early stages (stages 20-21) of chick limb development when positional values along the anterior-posterior (A-P) axis are being specified, GHox-8 is expressed in high amounts in the anterior mesoderm of the wing bud. Little expression of the gene is detectable in the middle region of the wing bud mesoderm or in the posterior mesoderm that contains the zone of polarizing activity, which is thought to be the source of a diffusible morphogen, possibly retinoic acid, that specifies the A-P positional values of the skeletal elements of the limb according to its local concentration. Similarly, at later stages of development (stages 23-25), high expression of GHox-8 is localized to the proximal anterior periphery of the wing bud, with no detectable expression in the proximal dorsal and ventral (myogenic) regions, or in the chondrogenic central core. In the proximal posterior periphery of the wing bud at these later stages of development, expression of GHox-8 is limited to a small region in the mid-proximal periphery corresponding to the posterior necrotic zone in which programmed cell death is occurring. The possible involvement of GHox-8 in programmed cell death during limb development is also suggested by the fact that it is expressed in the necrotic interdigital mesenchyme in 6-7 day (stage 31-32) wing buds.(ABSTRACT TRUNCATED AT 400 WORDS)

The expression pattern of the Distal-less homeo☐-containing gene Dlx-5 in the developing chick limb bud suggests its involvement in apical ectodermal ridge activity, pattern formation, and cartilage differentiation

Here we report the isolation from a chick limb bud cDNA library of a cDNA that contains the full coding sequence of chicken Dlx-5, a member of the Distal-less (Dlx) family of homeobox-containing genes that encode homeodomains highly similar to that of the Drosophila Distal-less gene, a gene that is required for limb development in the Drosophila embryo. The expression pattern of Dlx-5 in the developing chick limb bud suggests that it may be involved in several aspects of limb morphogenesis. Dlx-5 is expressed in the apical ectodermal ridge (AER) which directs the outgrowth and patterning of underlying limb mesoderm. During early limb development Dlx-5 is also expressed in the mesoderm at the anterior margin of the limb bud and in a discrete group of mesodermal cells at the mid-proximal posterior margin that corresponds to the posterior necrotic zone. These mesodermal domains of Dlx-5 expression roughly correspond to the anterior and posterior boundaries of the progress zone, the group of highly proliferating undifferentiated mesodermal cells underneath the AER that will give rise to the skeletal elements of the limb and associated structures. The AER and anterior and posterior mesodermal domains of Dlx-5 expression are regions in which the homeobox-containing gene Msx-2 is also highly expressed, suggesting that Dlx-5 and Msx-2 might be involved in regulatory networks that control AER activity and demarcate the progress zone. In addition, Dlx-5 is expressed in high amounts by the differentiating cartilaginous skeletal elements of the limb, suggesting it may be involved in regulating the onset of limb cartilage differentiation.

Gap junctional communication during limb cartilage differentiation

The onset of cartilage differentiation in the developing limb bud is characterized by a transient cellular condensation process in which prechondrogenic mesenchymal cells become closely apposed to one another prior to initiating cartilage matrix deposition. During this condensation process intimate cell-cell interactions occur which are necessary to trigger chondrogenic differentiation. In the present study, we demonstrate that extensive cell-cell communication via gap junctions as assayed by the intercellular transfer of lucifer yellow dye occurs during condensation and the onset of overt chondrogenesis in high density micromass cultures prepared from the homogeneous population of chondrogenic precursor cells comprising the distal subridge region of stage 25 embryonic chick wing buds. Furthermore, in heterogeneous micromass cultures prepared from the mesodermal cells of whole stage 23/24 limb buds, extensive gap junctional communication is limited to differentiating cartilage cells, while the nonchondrogenic cells of the cultures that are differentiating into the connective tissue lineage exhibit little or no intercellular communication via gap junctions. These results provide a strong incentive for considering and further investigating the possible involvement of cell-cell communication via gap junctions in the regulation of limb cartilage differentiation.

Cartilage proteoglycan core protein gene expression during limb cartilage differentiation

Changes in the steady-state cytoplasmic levels of mRNA for the core protein of the major sulfated proteoglycan of cartilage were examined during the course of limb chondrogenesis in vitro using cloned cDNA probes. Cytoplasmic core protein mRNA begins to accumulate at the onset of overt chondrogenesis in micromass culture coincident with the crucial condensation phase of the process, in which prechondrogenic mesenchymal cells become closely juxtaposed prior to depositing a cartilage matrix. The initiation of core protein mRNA accumulation coincides with a dramatic increase in the accumulation of mRNA for type II collagen, the other major constituent of hyaline cartilage matrix. Following condensation, there is a concomitant progressive increase in cytoplasmic core protein and type II collagen mRNA accumulation which parallels the progressive accumulation of cartilage matrix by the cells. The relative rate of accumulation of cytoplasmic type II collagen mRNA is greater than twice that of core protein mRNA during chondrogenesis in micromass culture. Cyclic AMP, an agent implicated in the regulation of chondrogenesis elicits a concomitant two- to fourfold increase in both cartilage core protein and type II collagen mRNA levels by limb mesenchymal cells. Core protein gene expression is more sensitive to cAMP than type II collagen gene expression. These results suggest that the cartilage proteoglycan core protein and type II collagen genes are coordinately regulated during the course of limb cartilage differentiation, although there are quantitative differences in the extent of expression of the two genes.

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. Proximal phalanges are duplicated in Has2 mutant limbs indicating an involvement of HA in patterning specific portions of the digits. The growth plates of Has2-deficient skeletal elements are severely abnormal and disorganized, with a decrease in the deposition of aggrecan in the matrix and a disruption in normal columnar cellular relationships. Furthermore, there is a striking reduction in the number of hypertrophic chondrocytes and in the expression domains of markers of hypertrophic differentiation in the mutant growth plates, indicating that HA is necessary for the normal progression of chondrocyte maturation. In addition, secondary ossification centers do not form in the central regions of Has2 mutant growth plates owing to a failure of hypertrophic differentiation. In addition to skeletal defects, the formation of synovial joint cavities is defective in Has2-deficient limbs. Taken together, our results demonstrate that HA has a crucial role in skeletal growth, patterning, chondrocyte maturation and synovial joint formation in the developing limb.

Temporal and spatial analysis of cartilage proteoglycan core protein gene expression during limb development by in situ hybridization

As limb mesenchymal cells differentiate into chondrocytes they initiate the synthesis of a cartilage-specific sulfated proteoglycan, cartilage-characteristic type II collagen, and other cartilage-specific proteins. In the present study, in situ hybridization with a 32P-labeled cloned cDNA probe complementary to mRNA encoding the core protein of cartilage proteoglycan has been used to visualize and localize the accumulation of cartilage proteoglycan core protein mRNA sequences during development of the chick limb bud in vivo. When the probe was hybridized to sections through 7-day (stage 32) limbs, an intense hybridization signal was observed over the well-differentiated cartilage rudiments of the limb, while no signal above background was observed over nonchondrogenic tissues including muscle, loose connective tissue, and epidermis. At early stages of limb development, an accumulation of silver grains representing hybridizable core protein mRNA first became detectable in the proximal central core of the limb where the prechondrogenic condensation of mesenchymal cells that characterizes the onset of cartilage differentiation was occurring. In fact, the pattern of silver grain accumulation closely followed the pattern of mesenchymal cell condensation, and no hybridizable core protein mRNA sequences were detectable in the limb bud prior to condensation. Cartilage-characteristic type II collagen mRNA was colocalized with core protein mRNA in the condensing central core of the limb suggesting that the genes for these two major constituents of cartilage matrix are coordinately regulated at the onset of chondrogenesis. Furthermore, the appearance of hybridizable core protein mRNA was closely followed by the appearance of the protein for which it codes as detected by immunohistochemical staining with monospecific antibody. These observations support the hypothesis that at the initial stages of limb chondrogenesis core protein gene expression is controlled primarily at the transcriptional level.

Temporal and spatial distribution of fibronectin during development of the embryonic chick limb bud

Indirect immunofluorescence has been used to study the distribution of fibronectin during the course of embryonic chick limb morphogenesis and differentiation. At all stages of development from 19 through 25, fibronectin is distributed throughout the non-differentiating mesenchymal tissue directly subjacent to the apical ectodermal ridge (AER), i.e. the mesenchyme extending 0.15 mm or so from the AER. Fibronectin is also distributed throughout the proximal condensing central core of the limb during the early stages of cartilage differentiation. In fact, fibronectin persists as a major component of the intercellular matrix in the central core of the limb following overt chondrogenic differentiation, since it is present as late as stage 27 throughout the well-differentiated cartilage rudiments of the radius, ulna, and humerus. The detection of fibronectin in differentiated cartilage is facilitated by pre-treatment of the sections with testicular hyaluronidase prior to immunofluorescent staining. In contrast to the chondrogenic central core of the limb, in the peripheral dorsal and ventral (myogenic) regions in which muscle differentiation is progressing, there is a progressive and striking diminution in fibronectin staining. By stage 27, little, if any, is present in the well-differentiated muscle primordia. Finally, at all stages of development, there is an accumulation of fibronectin at the ectodermal-mesenchymal interface, suggesting it is a component of embryonic limb basement membranes. On the basis of these observations, the possible role of fibronectin in limb cartilage and muscle differentiation and in other aspects of limb morphogenesis is discussed.

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 Col1a1 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. In situ hybridization studies indicated that Dlx5 is expressed in chick calvarial osteoblasts (cCOB) in vivo. Northern blot analysis indicated that Dlx5 expression in cultured cCOBs is induced concurrently with osteoblastic markers. To study the effect of overexpression of Dlx5 on osteoblast differentiation, we infected primary osteoblast cultures from 15‐day‐old embryonal chicken calvaria with replication competent retroviral vectors [RCASBP(A)] expressing Dlx5 or control replication competent avian splice acceptor brianhightiter polymerase subtype A [RCASBP(A)]. Expression of Col1a1, osteopontin, alkaline phosphatase, and osteocalcin messenger RNA (mRNA) occurred sooner and at higher levels in cultures infected with RCASBP(A)DLX5 than in RCASBP(A)‐infected cultures. Mineralization of Dlx5‐expressing cultures was evident by days 12‐14, and RCAS‐infected control osteoblasts did not begin to mineralize until day 17. Dlx5 also stimulated osteoblastic differentiation of calvarial cells that do not normally undergo osteoblastic differentiation in vitro. Our results suggest that Dlx5 plays an important role in inducing calvarial osteoblast differentiation.