Rebecca S. Reiter

Cloning and characterization of a novel bicoid-related homeobox transcription factor gene, RIEG, involved in Rieger syndrome

Rieger syndrome (REG) is an autosomal–dominant human disorder that includes anomalies of the anterior chamber of the eye, dental hypoplasia and a protuberant umbilicus. We report the human cDNA and genomic characterization of a new homeobox gene, RIEG, causing this disorder. Six mutations in RIEG were found in individuals with the disorder. The cDNA sequence of Rieg, the murine homologue of RIEG, has also been isolated and shows strong homology with the human sequence. In mouse embryos Rieg mRNA localized in the periocular mesenchyme, maxillary and mandibular epithelia, and umbilicus, all consistent with RIEG abnormalities. The gene is also expressed in Rathkes pouch, vitelline vessels and the limb mesenchyme. RIEG characterization provides opportunities for understanding ocular, dental and umbilical development and the pleiotropic interactions of pituitary and limb morphogenesis.

Stage-related capacity for limb chondrogenesis in cell culture☆

Cells from wing buds of varying-stage chick embryos were dissociated and grown in culture to test their capacity for cartilage differentiation. Micro-mass cultures were initiated with a cell layer greater than confluency, which occupied a restricted area of the culture dish surface (10–13 mm2). Cells from stage 24 chick embryo wing buds (prior to the appearance of cartilage in vivo) undergo cartilage differentiation in such cultures. Typically, during the first 1–2 days of culture, cells form aggregates (clusters of cells with a density 1.5 times greater than that of the surrounding nonaggregate area). By Day 3, virtually all aggregates differentiate into cartilage nodules which are easily recognized by their Alcian blue staining (pH 1.0) extracellular matrix. Subsequently, nodules increase in size, and adjacent nodules begin to coalesce. Micro-mass cultures were used to test the chondrogenic capacity of wing bud cells from chick embryos representing the different stages of limb development up to the appearance of cartilage in vivo (stages 17–25). Cells from embryo stages 21–24 form aggregates which differentiate into cartilage nodules in vitro with equal capacity (scored as number of nodules per culture). In contrast, cells from embryo stages 17–19 form aggregates in similar numbers, but these aggregates never differentiate into nodules under routine conditions. However, aggregates which form in cultures of stage 19 wing bud cells do differentiate into cartilage nodules if exposed to dibutyryl cyclic AMP and theophylline. Cells from stage 20 embryos manifest a varying capacity to form cartilage nodules; apparently, this is a transition stage. Cells from stage 25 embryos produce cartilage in vitro without forming either aggregates or nodules. Based on the results presented in this paper, the authors propose a model for cartilage differentiation from embryonic mesoderm cells involving: (1) aggregation, (2) acquisition of the ability to respond to the environment in the aggregate, (3) elevated intracellular cyclic AMP levels, and (4) stabilization and expression of cartilage phenotype.

A novel homeobox gene PITX3 is mutated in families with autosomal-dominant cataracts and ASMD

We report here the identification of a new human homeobox gene, PITX3, and its involvement in anterior segment mesenchymal dysgenesis (ASMD) and congenital cataracts in humans. The PITX3 gene is the human homologue of the mouse Pitx3 gene and is a member of the RIEG/PITX homeobox gene family. The protein encoded by PITX3 shows 99% amino-acid identity to the mouse protein, with 100% identity in the homeodomain and approximately 70% overall identity to other members of this family. We mapped the human PITX3 gene to 10q25 using a radiation-hybrid panel. A collection of 80 DNA samples from individuals with various eye anomalies was screened for mutations in the PITX3 gene. We identified two mutations in independent patients. A 17-bp insertion in the 3´-end of the coding sequence, resulting in a frame shift, occured in a patient with ASMD and cataracts, and a G→A substitution, changing a codon for serine into a codon for asparagine, in the 5´-end of the gene occured in a patient with congenital cataracts. Both mutations cosegregate with the disease phenotype in families, and neither were found in up to 300 control individuals studied. Further expression analysis of Pitx3in the mouse supports a unique role in early ocular development, with later expression extending to the midbrain, tongue, incisors, sternum, vertebrae and limbs. These data strongly suggest a role for PITX3 in ASMD and cataracts and provide new evidence of the contribution of the RIEG/PITX gene family to the developmental program underpinning normal eye formation.

The influence of epithelia on cartilage and loose connective tissue formation by limb mesenchyme cultures

Abstract In order to explain the observation that normally nonchondrogenic limb mesenchyme forms extensive cartilage in culture, the possibility that limb ectoderm inhibits chondrogenesis is examined. Small pieces of quail or chick ectoderm are grafted onto micromass cultures of wing mesenchyme from stage 23–34 chick embryos. The presence of nonridge wing or several other epithelia results in increased collagen accumulation in the underlying mesenchyme, a delay in cell differentiation, and the eventual formation of loose connective tissue, as determined by transmission electron microscopy. The influence can occur across Nuclepore filters having 0.1-μm-diameter pores and ultrathin Millipore filters, but not across Millipore filters of standard thickness. The influence is not contact dependent since cell processes do not cross these filters. The apical ectodermal ridge has the additional effect of stimulating mesenchymal outgrowth. These results support the idea that the ectoderm plays a direct but negative role in the formation of a chondrogenic core within the developing limb.

A TISSUE CULTURE ANALYSIS OF THE STEPS IN LIMB CHONDROGENESIS

SummaryTissue-culture methods can be used to test the developmental capacity of embryonic cells. In micro-mass cultures, derived from wing cells of stages 21 through 24 chick embryos, aggregates of cells form and then differentiate into cartilage nodules, as judged by the presence of an Alcian blue staining extracellular matrix. Wing cells derived from embryos as young as stage 17 can form aggregates. However, unless they are treated with db cyclic AMP and theophylline, it is not until stage 20 that these aggregates can produce cartilage in culture. In clonal cell culture, cartilage colonies are not produced by primary cell suspensions of limb cells until stage 25 when overt cartilage differentiation is occurring in vivo. It is possible to obtain clonable cartilage cells from limb cells from embryos between stages 20 and 24 if the cells are either treated with db cyclic AMP and theophylline or maintained in suspension culture for 12 to 48 hr. On the basis of these in vitro results a multiple step model for the conversion of limb mesenchyme into cartilage cells is proposed. The model involves the appearance of cells with a predisposition to form aggregates, development of the capacity to form cartilage in response to elevated levels of cyclic AMP, the appearance of receptors that translate changes in either cell shape or cell cycle parameters into elevated levels of cyclic AMP, aggregation, elevated levels of cyclic AMP, cartilage cell determination, and differentiation. This model can serve as the basis for further tests.

Transient expression of a cell surface heparan sulfate proteoglycan (syndecan) during limb development

Syndecan is an integral membrane proteoglycan that contains both heparan sulfate and chondroitin sulfate chains and that links the cytoskeleton to interstitial extracellular matrix components, including collagen and fibronectin. Immunohistochemistry with a monoclonal antibody directed to the core protein of the syndecan ectodomain has been used to analyze the distribution of this proteoglycan in the developing mouse limb bud and in high-density cultures of limb mesenchyme cells. By Day 9 of gestation when the limb buds are just apparent, syndecan is detected on cells throughout the limb region, including both ectodermal and mesenchymal components. This distribution does not change as the limb bud elongates along its proximodistal axis, except for its reduction in the apical ectodermal ridge. By Day 11, the intensity of immunofluorescence in the central core decreases relative to other regions. By Day 13 immunostaining is lost in the regions destined for chondrogenesis and myogenesis but persists in the limb ectoderm and peripheral and distal mesenchyme. In the limb mesenchyme cell cultures, syndecan is initially undetected, but is found throughout the culture by 24 hr. With further culture the antigen becomes reduced in chondrogenic foci and in association with myogenic cells. When chick limb ectoderm is placed on the high-density cultures, immunoreactivity in the mouse mesenchyme is enhanced suggesting that epithelial-mesenchymal interactions modulate syndecan expression in the limb bud. Based on analysis of 35S-labeled syndecan from the cultures, syndecan from limb mesenchyme cells contains more glycosaminoglycan chains and is larger in size than the previously described polymorphic forms of syndecan from various epithelia. The high affinity of syndecan for components of the extracellular matrix and its distribution in the early limb bud are consistent with a role in maintaining the morphologic integrity of the limb bud during the period of initiation and rapid outgrowth, and in preventing the onset of chondrogenesis.

Depression by hyaluronic acid of glycosaminoglycan synthesis by cultured chick embryo chondrocytes.

Abstract The effects of hyaluronic acid on the expression of differentiation by pure populations of cultured chick embryo chondrocytes are studied. In medium containing serum, hyaluronic acid has little effect on sulfate incorporation. In the absence of serum, 200 μg/ml of hyaluronic acid inhibits sulfate incorporation into hyaluronidase-sensitive material associated with the cell layer by as much as 50%. This effect is seen by 6 hr and is progressive. Hyaluronic acid has no effect on leucine or thymidine incorporation or sulfate uptake, matrix turnover, or collagen synthesis under these same conditions. It is shown that hyaluronic acid inhibits chondroitin sulfate synthesis and the accumulation of newly synthesized proteoglycan in the cell layer. These effects are not produced by other glycosaminoglycans, such as chondroitin sulfate, keratan sulfate, or heparan sulfate, or by monosaccharides, but oligosaccharides derived from hyaluronic acid mimic the effect of hyaluronic acid. These data indicate that specific glycosaminoglycans, such as hyaluronic acid, can affect the synthesis and accumulation of other glycosaminoglycans and should be considered as possible regulators of specific cellular activities.

Stage- and position-related changes in chondrogenic response of chick embryonic wing mesenchyme to treatment with dibutyryl cyclic AMP

The responsiveness of high-density cell cultures (micromass) prepared from different stages or different regions of chick embryonic wings to treatment with dibutyryl cyclic AMP (dbcAMP) and related compounds is used to define cellular heterogeneity and steps in cartilage differentiation. Cultures of stage 19 wing mesenchyme form aggregates (areas of greater cell density than adjacent areas), but not cartilage nodules. Treatment with dbcAMP (1 mM) results in the aggregates differentiating into cartilage nodules. Cultures from stage 21–24 wings form aggregates which spontaneously differentiate into cartilage nodules. Treatment with dbcAMP or cholerotoxin for only 12 hr results in the formation of a continuous sheet of cartilage, as defined by Alcian blue stainign, 35SO42− autoradiography, and immunofluorescence with antibodies to cartilage-specific proteoglycan. The peripheral, normally nonchondrogenic region of the stage 24 limb and the distal 0.5 mm of the stage 24–26 wing in micromass culture respond to dbcAMP similarly of the whole stage 24 limb. On the other hand, cultures from whole wings or cross-sectional slices from the midregion of stage 26 wings behave like stage 19 wings, except for the additional presence of small scattered clusters of cartilage cells. These results suggest the existence of at least four subpopulations of limb mesenchyme cells (non-chondrogenic cells, dbcAMP-responsive aggregate cells, dbcAMP-responsive nonaggregate cells, and spontaneously chondrogenic cells). The proportions of cells having these properties change temporally and spatially in a manner which is correlated with cartilage histogenesis.

Role of VEGF family members and receptors in coronary vessel formation

The specific roles of vascular endothelial growth factor (VEGF) family members and their receptors (VEGFRs) in coronary vessel formation were studied. By using the quail heart explant model, we found that neutralizing antibodies to VEGF‐B or VEGF‐C inhibited tube formation on the collagen gel more than anti–VEGF‐A. Soluble VEGFR‐1, a receptor for VEGF‐A and ‐B, inhibited tube formation by 87%, a finding consistent with that of VEGF‐B inhibition. In contrast, addition of soluble VEGFR‐2, a receptor for VEGF family members A, C, D, and E, inhibited tube formation by only 43%. Acidic FGF‐induced tube formation dependency on VEGF was demonstrated by the attenuating effect of a soluble VEGFR‐1 and ‐2 chimera. The localization of VEGF R‐2 and R‐3 was demonstrated by in situ hybridization of serial sections, which documented marked accumulations of transcripts for both receptors at the base of the truncus arteriosus coinciding with the temporal and spatial formation of the coronary arteries by means of ingrowth of capillary plexuses. This finding suggests that both VEGFR‐2 and R‐3 may play a role in the formation of the coronary artery roots. In summary, these experiments document a role for multiple members of the VEGF family and their receptors in formation of the coronary vascular bed.

Position-related capacity for differentiation of limb mesenchyme in cell culture.

Abstract A quantitative comparison (i.e., number of cartilage nodules) of cartilage differentiation was made between micromass cell cultures prepared with cells from different locations (core vs periphery) within prechondrogenic chick wing buds. Wing bud core cells in micromass culture exhibit a greater developmental bias toward cartilage differentiation than periphery cells from the same limbs. In addition, myogenic cells appear more frequently in cultures prepared from wing bud periphery than in those prepared from core tissue. Therefore a stage 23–24 wing bud is not a homogeneous population of multipotential mesenchymal cells. Instead, a stage 23–24 wing bud contains two classes of cells, each characterized by a bias for either cartilage or muscle differentiation, and a third class of uncharacterized mesenchymal cells.