Caroline N.D. Coelho

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)

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.

A gradient of gap junctional communication along the anterior-posterior axis of the developing chick limb bud

A modification of the scrape-loading/dye transfer technique was used to study gap junctional communication along the anterior-posterior (A-P) axis of embryonic chick wing buds at an early stage of development (stage 20/21) when positional values along the A-P axis are being specified. Extensive intercellular transfer of the gap junction-permeable dye, lucifer yellow, from scrape-loaded mesenchymal cells to contiguous cells occurs in the posterior mesenchymal tissue of the wing bud adjacent to the zone of polarizing activity, which is thought to be the source of a diffusible morphogen that specifies A-P positional identity according to its local concentration. Considerably less transfer of lucifer yellow dye occurs in scrape-loaded mesenchymal tissue in the middle of the limb bud compared to posterior mesenchymal tissue, and little or no transfer of lucifer yellow is observed in the mesenchymal tissue in the anterior portion of the limb bud. No intercellular transfer of the gap junction-impermeable dye, rhodamine dextran, occurs in any region of the limb bud. These results indicate that there is a gradient of gap junctional communication along the A-P axis of the developing chick wing bud. This gradient of gap junctional communication along the A-P axis might generate a graded distribution of a relatively low molecular weight intracellular regulatory molecule involved in specifying A-P positional identities.

Type IX collagen gene expression during limb cartilage differentiation

Changes in the steady-state levels of mRNAs for the alpha 1(IX) and alpha 2(IX) polypeptide chains of cartilage-characteristic type IX collagen were examined during the course of chick limb chondrogenesis in vitro and in vivo. Cytoplasmic type IX collagen mRNAs begin to accumulate at the onset of overt chondrogenesis in high density 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 type IX collagen mRNA accumulation at condensation coincides with the initiation of accumulation of cartilage proteoglycan core protein mRNA and with a striking increase in type II collagen mRNA accumulation. Following condensation in vitro, there is a concomitant progressive increase in cytoplasmic type IX collagen, core protein, and type II collagen mRNA levels which parallels the progressive accumulation of cartilage matrix. Type IX collagen mRNAs also begin to accumulate at the initiation of overt chondrogenesis in vivo in the chondrogenic central core of the developing limb bud. In contrast, little, or no type IX collagen mRNAs are detectable in the nonchondrogenic peripheral regions of the developing limb bud.

Role of the Transforming Growth Factor- β (TGF- β ) Family, Extracellular Matrix, and GAP Junctional Communication in Limb Cartilage Differentiation

The differentiation of limb mesenchymal cells into chondrocytes involves a sequential series of regulatory events mediated in part by extracellular matrix macromolecules, peptide growth factors, cell-cell and cell-matrix interactions, cytoskeletal reorganization, and intracellular second messengers such as cAMP. In the present manuscript we describe some of our current studies on the role of and the possible relationship between the TGF-β family of growth factors and extracellular matrix and cell surface molecules including the membrane-intercalated proteoglycan, syndecan in the regulation of chick limb cartilage differentiation. We also describe studies indicating that intercellular communication via gap junctions may be involved in the regulation of chondrogenesis.

Homeobox-Containing Genes and GAP Junctional Communication in Pattern Formation during Chick Limb Development

Elucidating the mechanisms involved in eliciting the formation of the various skeletal elements of the limb in their appropriate position and sequence along the anterior-posterior (A-P) axis is fundamental to understanding normal and abnormal vertebrate limb development. The zone of polarizing activity (ZPA) at the posterior margin of the developing limb bud appears to be the source of a diffusible morphogen, possibly retinoic acid, that becomes distributed in a graded fashion across the A-P axis of the limb, and specifies the A-P positional values of the skeletal elements of the limb according to its local concentration (Brickell and Tickle, 1989; Eichele, 1989). Homeobox-containing genes have been implicated in the regulation of pattern formation during development, and several homeobox genes exhibit spatially-restricted and temporally-regulated patterns of expression during vertebrate limb development (Oliver et al., 1988, 1989; Dolle et al., 1989; Hill et al., 1989; Robert et al., 1989; Wedden et al., 1989). In the present manuscript we describe some of our current studies on the isolation and characterization of several homeobox genes expressed during embryonic chick limb development, and present evidence indicating that some of these genes are expressed in a graded fashion along the A-P axis of the chick limb bud as positional values along the A-P axis are being specified. We also report on studies indicating that intercellular signalling via gap junctions may be involved in specification of pattern along the A-P axis of the chick limb bud.