The tandemly repeated sequences of cartilage link protein contain the sites for interaction with hyaluronic acid.

The ternary complex involving link protein (LP), proteoglycan monomer, and hyaluronic acid (HA) is an important component of the extracellular matrix of cartilage. LP contains tandemly repeated sequences that were tested for their ability to interact with HA. A solid-phase assay was developed in which LP could specifically bind to immobilized HA. Detection of LP was by means of an antiserum directed against a peptide from the NH2-terminal half of LP. LP binding to HA could be inhibited with mAb 8A4 (Caterson, B., J. R. Baker, J. E. Christner, Y. Lee, and M. Lentz. 1985. J. Biol. Chem. 260:11348-11356). Using synthetic peptides that correspond to specific amino acid sequences of chicken LP (Deák, F., I. Kiss, K. J. Sparks, W. S. Argraves, G. Hampikian, and P. F. Goetinck. 1986. Proc. Natl. Acad. Sci. USA. 83:3766-3770) the epitopes for mAb 8A4 were determined to reside in peptides Gly217-Pro226 and Arg316-Arg325. These two peptides were also capable of inhibiting the interaction between LP and HA. The peptide Trp242-Val251 and Pro339-Val348 could also inhibit the interaction between LP and HA. All four peptides reside in the tandemly repeated domains of LP and they contain clusters of positively charged amino acids. Polylysine could not inhibit the interaction of LP with HA. The results indicate that the sites for interaction with HA are in the tandemly repeated sequences of LP and that there are four potential sites available for that interaction.

Newt squamous carcinoma proves phylogenetic conservation of tumors as caricatures of tissue renewal.

Although regeneration-competent newts like Notophthalmus viridescens have been reported to be resistant to carcinogenesis, we have been able to induce transplantable epidermal squamous cell carcinomas with 10-20% incidence by implanting 20-methylcholanthrene s.c. into the scapular region, a tissue that cannot regenerate. As soon as 1 week after exposure to this carcinogen, malignant cells were present in the treated skin, and after 4 weeks, macroscopic tumors of infiltrating squamous carcinoma cells positive for Type IV collagenase and/or rasHa p21 had dissolved areas of the epidermal basement membrane and colonized the dermis. Analysis of Ki-67 expression revealed that these tumors grow via a high growth fraction rather than a short cell cycle time. Morphological and immunohistochemical analyses showed that these tumors caricature the biology of the renewing epidermis: the presence of basal-like cells; differentiating cells; apoptotic cells; and keratinized horn pearls with an exaggerated or overabundant stem cell compartment as compared to the differentiated cell compartment. Immunochemical analyses indicated that the squamous carcinomas arose from the epidermis rather than the mucous glands. Thus, the principle that malignant tumors caricature the process of tissue renewal originally established in rodent tumors appears to be valid down the phylogenetic tree at least to regeneration-competent amphibia. Such a broad conservation indicates that the caricature principle also holds in human tumors.

Graded expression of Emx-2 in the adult newt limb and its corresponding regeneration blastema.

Amputation of a newt limb causes stump cells to organize the reformation of the missing structures. The phenomenon is remarkably precise in that the regeneration is perfect. During the first few days following amputation, the tissue proximal to the plane of amputation gives rise to the blastema, an area of growth composed of mesenchymal cells covered by a single epithelium. The blastema possesses a morphogenetic potential characteristic of the structures that have been amputated. Looking for control genes putatively involved in regeneration, we cloned the newt version of the mouse and human Emx-2. Its expression is restricted to the skin of the regeneration territories and is graded along the proximal-distal axis of both forelimb and hindlimb, with higher levels in distal regions. The regeneration blastema also show this proximal-distal graded level of expression with distal blastemas (mid-radius and ulna) showing higher levels of expression when compared to blastemas of more proximal origin (mid-humerus). Finally, retinoic acid proximalizes both the level of Emx-2 expression and the positional memory of the blastema suggesting Emx-2 may participate in pattern formation by specifying positional information.

Mononuclear leukocytes in the newt limb blastema: in vitro behavior.

Since the precise interactions of the various newt limb blastema cell types (pleiomorphic, bipolar, signet and multinucleated cells) with one another are difficult to ascertain in vivo, in this study we describe the in vitro behavior and interactions of these cell types with one another. The data show that the mononuclear signet cells interact (fuse) with other signet cells or fuse directly with the multinucleated cells. Further, they indicate that the signet cells are in fact mononuclear leukocytes and the giant cells are osteoclasts. Therefore, we conclude that these two cell types are not formed by dedifferentiation but are of hematopoietic origin and may play minor roles in blastema production.

Role of 5' HoxD genes in chondrogenesis in vitro.

In the present study, we have examined by in situ hybridization, expression of five 5' HoxD cluster genes (D9, D10, D11, D12 and D13) during chondrogenesis of chick limb bud mesenchymal cells in vitro. After one day in culture, D9 and D13 gene expression was restricted to patches of mesenchymal cells, while expression of D10, Dll, and D12 gene was prominent in all mesenchymal cells. In 3-day cultures, D9 and D13 genes were expressed only in cartilage nodules, while D10, Dll, and D12 genes were expressed in both cartilage nodules and in all mesenchymal cells. These observations indicate two different patterns of expression; one for D9 and D13, and a different one for D10, Dll, and D12. These patterns of expression seem to correlate with patterns of cell proliferation and differentiation to chondrocytes. The role of these HoxD genes was further investigated by employing antisense S-oligomers. We found that oligodeoxynucleotides complementary to HoxD (D10-D 13) mRNAs were capable of inhibiting chondrogenesis. These data suggest that expression of HoxD genes is required for mesenchymal condensation, and differentiation to chondrocytes. This in turn implies that these HoxD genes aside from their role in the patterning of the developing skeletal elements might regulate down-stream factors necessary for cartilage differentiation as well.

Protein synthesis in the brain of newts undergoing limb regeneration.

The nervous system plays an important role during the process of amphibian limb regeneration. However, the molecules that are involved in such a control of regeneration are largely unknown. We have attempted to map protein synthesis in the brains of intact newts and from newts undergoing limb or tail regeneration. Our results show unique protein synthesis in the brain of newts undergoing limb regeneration. Such an analysis can lead to the identification and characterization of these proteins.

A unique aged human retinal pigmented epithelial cell line useful for studying lens differentiation in vitro.

Lens regeneration occurs in some urodeles and fish throughout their adult life. Such an event is possible by the transdifferentiation of the pigment epithelial cells (PECs) from the dorsal iris. Studies of this event at the cellular level have been facilitated owing to the ability of PECs to become lens cells even when they are placed in culture, outside of the eye. In fact, PECs possess the capacity for transdifferentiation regardless of the origin of species or age. However, studies at the molecular level are still hindered by the intrinsic problems of primary cultures, namely storage, reproducibility and genetic manipulation. In an attempt to establish an ideal model system for lens transdifferentiation, we have analyzed the ability of a human dedifferentiated PEC line to differentiate into lens. We have found that this cell line can indeed be induced to synthesize crystallin and morphologically differentiate to three-dimensional structures resembling lentoids under controlled treatment in vitro. Gene expression studies also provided important insights into the role of key genes. This human cell line can be used for detailed genetic studies in order to identify the key factors involved in lens transdifferentiation from PECs.

9-cis retinoic acid antagonizes the stimulatory effect of 1,25 dihydroxyvitamin D3 on chondrogenesis of chick limb bud mesenchymal cells: interactions of their receptors.

Retinoids or vitamin D have been found to profoundly affect pattern formation and chondrogenesis in the developing limb. These substances mediate their actions through their nuclear receptors. In the present investigation, we present data showing that 9-cis RA, the ligand for RXR can stimulate chondrogenesis of chick limb bud mesenchymal cells, however, in combination, it antagonizes the stimulatory effect of vitamin D in the same system. The receptors for 9-cis RA (RXR) and vitamin D (VDR) were also shown to be present in the mesenchymal cells and to form heterodimers. These results implicate these receptors in cartilage differentiation during limb development.

Can insights into urodele limb regeneration be achieved with cell cultures and retroviruses?

Recent advances in the field of amphibian limb regeneration have provided insights into its cellular and molecular events. This review summarizes the development of cell lines from limb tissues and their application to the study of transdifferentiation and limb regeneration. In addition, the availability of suitable retroviral vectors for salamanders is discussed for it has opened new avenues for experimentation at the molecular level.

Cloning of homoeobox sequences expressed in the intact and the regenerating newt eye.

In this report we present the sequences of four different homeoboxes cloned from the intact and regenerating eye of the newt Notophthalmus viridescens. All these homeoboxes are novel for this species.

Similarities between a conserved sequence element of homoeo boxes and other genes.

We report here the homology of different genes with an 18-nucleotide sequence element derived from a conserved region of the homoeo boxes. Possible evolutionary relationships are discussed.

Regulation of Prox 1 during lens regeneration.

PURPOSE: To determine the expression pattern of Prox 1 during the process of lens regeneration in the urodele Notophthalmus viridescens. METHODS: Polymerase chain reaction was performed to amplify a partial newt Prox 1 sequence. In situ hybridization and immunodetection methods were used to detect the Prox 1 mRNA and the Prox 1 protein, respectively. RESULTS: Prox 1 mRNA was present in the retina and in the lens (in the epithelium and bow region) of the intact eye. Prox 1 protein was found to be predominantly present in the lens and dorsal iris of the intact eye, although some trace levels of Prox 1 protein were detected in the ventral iris as well. After lentectomy, expression of the mRNA was also pronounced in the dorsal dedifferentiating iris and the regenerating lens. The ventral iris also expressed Prox 1 but seemingly at lower levels. Although Prox 1 protein showed upregulation in the dorsal iris during the process of lens regeneration, trace levels were also detected in the ventral iris. In the retina, Prox 1 protein was distributed in horizontal cells of the inner nuclear layer, whereas the mRNA was expressed in all layers of the retina. CONCLUSIONS: Prox 1 was unevenly distributed in the intact cells of the newt iris, with significantly higher levels of Prox 1 protein present in the dorsal versus the ventral margin. This protein was differentially regulated during the process of lens regeneration, with obvious upregulation in the dorsal iris. Prox 1 is the first transcriptional factor to be shown to be regulated in the dorsal versus ventral iris during the process of lens regeneration.

Abnormal codon recognition of glycyl-tRNA from the posterior silk glands of Bombyx mori.

Two species of glycine tRNA (tRNA1Gly and tRNA2Gly) were purified from the posterior silk glands of Bombyx mori. Digestion of tRNA1Gly wit RNase T1 revealed that this tRNA corresponds to the species which has recently been sequenced by Garel and Keith (1), and Züniga and Steitz (2), independently. However, the position 9 of our tRNA2Gly was occupied by unmodified guanosine instead of m1G. The codon recognition properties of highly purified tRNA1Gly and tRNA2Gly were investigated in detail according to the method of Nirenberg and Leder (3). All four glycine codons were recognized by tRNA1Gly and by tRNA2Gly. These results suggest the presence of abnormal base-pairs between the first base G of the anticodon and all the third bases of the codons, or the recognition by "two-out-of-three" type reading.

Protein separation techniques in the study of tissue regeneration.

The present report reviews the use of protein separation by means of two-dimensional gel electrophoresis in the study of tissue regeneration. It is shown that such an approach can provide data on protein synthesis in different stages of limb regeneration or comparative data with other regenerative processes such as tail and lens regeneration. Such an approach is more realistic than other methods employing gene cloning or generation of antibodies and can lead to the actual identification and characterization of factors that are involved in these phenomena.

Embryogenesis and carcinogenesis: order and disorder.

Similarities and differences between embryogenesis and carcinogenesis are outlined. Recent advances on specific gene expression and developmental phenomena related to cancer are presented.

Effects of carcinogens on regenerating and non-regenerating limbs in amphibia (review).

The effects of chemical carcinogens on regenerating and non-regenerating limbs in amphibia are reviewed. The regeneration-competent tissues show a remarkable resistance to chemical carcinogenesis. However, chemical carcinogens can induce teratogenesis when they are administered to the regeneration blastema, while normal differentiation occurs in limited cases when they are administered to the intact limbs. The regenerative capacity may be considered as the deterrent to neoplasia in these tissues.

Lack of cellular differentiation with neoplastic-like proliferation in the limbs of the newt Cynops pyrrhogaster.

We report a case of a neoplastic-like proliferation of epidermal cells in the limbs of an adult newt. The undifferentiated cell mass infiltrated the mesodermal tissues. Possible effects of this event on limb regeneration are discussed.

Amphibian limb regeneration.

The Field of amphibian limb regeneration is reviewed. Molecular biology techniques are beginning to explain old experiments and to provide the means of a better understanding of this phenomenon. The major experimental problems of the field are discussed.

Pattern formation: from theoretical models to molecular biology.

The field of pattern formation in biological systems is viewed, taking into consideration the major theoretical and experimental information. The basic idea which has emerged from the different theoretical models is that short range cell-to-cell interactions could manifest the hierarchy of patterns. These interactions could actually be governed by simple signals in the cell surface. In the second part the experimental evidence is examined, namely, the genes that control pattern formation in Drosophila. The products of these genes display a variety of characteristics ranging from being molecules at the cell surface to regulatory proteins in the nucleus. The hypothesis is presented that molecules in the cytoplasm could connect the two parts. According to this, signals at the cell surface and subsequent cell-to-cell interactions should be of primary importance in the mechanisms that underlie morphogenesis.

Histological analysis of forelimb regeneration in the California newt Taricha granulosa.

The regenerative ability of the forelimbs of the California rough-skinned newt, Taricha granulosa was determined and compared to the same ability of the adult Mexican axolotl, Ambystoma mexicanum. Forelimbs were amputated distally at the wrist and limbs removed at 1, 2, 3, 4, 5 and 6 weeks post-amputation were examined by histological analysis. Since vitamin A and its derivatives cause extreme changes in pattern formation in the regenerating amphibian limb, we decided to study the ability of retinoic acid to proximalize the distal amputation. Animals injected with RA displayed apparent proximalization of the distal amputation.