The mesenchymal cadherin-11 is expressed in restricted sites during the ontogeny of the rat brain in modes suggesting novel functions.

Cadherin-11 (Cad-11), a cell cell adhesion molecule belonging to the "classical type II" cadherin family, is a marker of the loosely connected and migratory cellular elements of the mesenchyme. Interestingly, by using in situ hybridization, regional high expression of cad-11 was seen in the brain as well as the spinal cord. We made the following observations in rat embryos and neonates: (1) cad-11 first appears at the lips of the open neural tube; (2) shortly after neural tube closure, cad-11 delineates boundaries in the fore- and midbrain while a metameric signal is detected in the rhombencephalon; (3) cad-11 expression is found in specific neuroepithelia and ependyma; (4) in the fetal developing brain, cad-11 transcripts are present during the formation of precise cortical layers, in various brain nucleus or subsets of nuclei and in circumventricular organs; (5) intense cad-11 expression is located at the point of optic nerve exit and entry; (6) cad-11 signal accompanies, in a spatio-temporal manner, the dynamics of cell migration in the cortex from lateral ganglionic eminence through the cortico-striatal sulcus. These data are discussed, and hypotheses for additional and novel properties for cad-11 are suggested.

Cadherin 11 expression marks the mesenchymal phenotype: towards new functions for cadherins?

Cadherin-11 (cad-11) belongs to the cell adhesion type II cadherin family, which seems to have different functions from the classic cadherin family. This study shows the overall pattern of cad-11 gene expression during rat embryonic development, from the pregastrula to very late embryonic stage. Cad-11 is the first cadherin found to be highly expressed in the dispersed and migrating mesenchymal cells that originate from the neuroectodermal neural crest cells and from the pre-chordal and paraxial mesoderm. A burst of cad-11 expression appears during the epithelial to mesenchymal transition, as observed by sclerotome formation. Cad-11 mRNAs were present in all mesenchymal cells throughout the embryo, regardless of their embryonic origin. A proximo-distal and antero-posterior gradient of cad-11 expression is seen in the limb buds, genitalia, and tail. As development proceeds, while all epithelium are negative, cad-11 is present in all mesenchymal cells involved in various morphogenetic events, such as the mesenchyme condensations during chondrogenesis and in the formation of sclera, cornea, naris, palate and meninges. Cad-11 was strongly expressed in mesenchyme during lung or kidney branching morphogenesis or the many epithelium to mesenchyme inductions that operate in the nasal septum, skin, vibrissae, teeth and various glands. High levels of cad-11 transcripts were also found in the dispersed cells of the hyaloid plexus in the vitreous body and in the invading mesenchyme within the trabeculae of the outflow tract of the heart. Cad-11 is thus specific to the mesenchymal phenotype whatever the stage of embryonic development.

Sequence and distribution of Xenopus laevis E-cadherin transcripts.

Cadherins are calcium-dependent adhesive glycoproteins implicated in histogenetic processes. In Xenopus laevis, the distribution of classical cadherins N, E, EP, XB and U has been determined by immunofluorescence labeling or by in situ hybridization. In this study, we report the full-length sequence of the E-cadherin cDNA. Comparison with the other cadherin sequences available indicates that Xenopus E-cadherin is as homologous to Xenopus EP-cadherin as to the chicken L-CAM and to the mammalian E-cadherin. Although Xenopus E-cadherin protein sequence exhibits many short conserved motifs present in other E-cadherins, it differs remarkably from the chicken L-CAM and the mammalian E-cadherin in its appearance after gastrulation. In situ hybridization data showed that E-cadherin transcripts are homogenously distributed in all differentiating epithelia from early tailbud to post-metamorphic stage. In contrast to mouse E-cadherin, Xenopus E-cadherin was not detected transiently in the nervous system during embryogenesis and in the post-metamorphic stages.

Molecular transitions accompanying growth of the axial musculature of Xenopus laevis.

In this paper we examine the expression of several cell adhesion molecules and muscle differentiation markers during larval development and growth of the axial musculature of Xenopus laevis. We have identified a small group of cells, located on the dorsal extremity of the myotome, which express very high levels of N-CAM and early muscle markers such as desmin and muscle myosin, but do not express later-stage markers of muscle development such as sarcomeric actinin. These cells are most probably muscle precursor cells; they may participate in the generation of new fibres in the myotomal muscles of Xenopus. On the basis of these observations we propose a model of myotomal development in Xenopus characterized by a spatial segregation between regions of the myotome where new fibres are generated and regions where myotubes mature. As during muscle differentiation in vivo we observe a sharp change in the profile of expression of cell adhesion molecules, we suggest that different adhesion receptors are involved in the generation of new muscle fibres and in the growth and differentiation of already existing ones.

N-cadherin transcripts in Xenopus laevis from early tailbud to tadpole.

Cadherins are Ca(++)-dependent cell adhesion molecules which play a key role in morphogenesis and histogenesis. Two mRNAs clones (8 and 9) corresponding to two N-cadherin pseudo-allelic genes are present in Xenopus laevis. We report here that these transcripts share a highly homologous coding region but diverge in the non-coding region. We have determined the pattern of N-cadherin expression at the mRNA level by in situ hybridization with a riboprobe complementary to the EC5 domain of Xenopus N-cadherin clone 8. This part of the sequence is the least conserved in the cadherin gene family, minimizing the risk of cross-hybridization to other cadherins. N-cadherin transcripts are not detectable in the first stages of development. Expression first appears in the neural plate and reaches its maximum level in the CNS at tailbud stage. From early tadpole, it diminishes, so that a very weak signal is detected in the premetamorphic frog brain. N-cadherin expression is not uniform within the CNS, with some areas such as the roof of the rhombencephalon and the olfactory bulbs expressing higher levels of the transcripts. N-cadherin is present in several mesodermal derivatives such as the notochord, the pronephros, and the heart. It is, however, virtually absent from the myotomes and appears in skeletal muscles at later stages of differentiation. All placodes express high levels of N-cadherin. The non-neural ectoderm and the endoderm are always negative. In the brain and the heart, high levels of hybridization are observed with probes corresponding to both copies of the N-cadherin pseudo-allelic genes in their 5' non-coding region, indicating that both alleles are transcribed.

Study of the function and regulation of liver N-CAM in Xenopus laevis.

The liver of Xenopus laevis is a unique exception in terms of the cell adhesion molecules (CAM) which it expresses. In most species, hepatocytes are characterized by the expression of the epithelial Ca(2+)-dependent CAM E-cadherin or of closely related variants of this molecule (e.g., L-CAM); in Xenopus liver, however, the levels of expression of epithelial cadherins is very low while a thyroxine-inducible isoform of N-CAM is expressed in postmetamorphic hepatocytes. Since Xenopus liver N-CAM is localized in regions of contact between hepatocytes, it has been proposed that it might be involved in mediating hepatocyte adhesion in this species. In this study, we demonstrate that N-CAM can indeed act as a functional adhesion molecule in the liver of Xenopus and that its expression is correlated with a number of profound morphological changes of this organ. After thyroxine treatment, hepatocytes are no longer organized in long loose cords but in compact lobules of cells. Furthermore, at the ultrastructural level, plasma membranes are in much closer proximity with the appearance of electron-dense material in areas of closer contact. We have established two novel culture systems for premetamorphic Xenopus hepatocytes as adherent and non-adherent cells, and we describe the induction of expression of N-CAM in these cells. Given the difference in the profile of adhesion molecules present in the liver of Xenopus and of other species, our results are discussed in view of the importance of the expression of a specific set of cell adhesion molecules in defining the development of homologous organs in different species.

Transdifferentiated embryonic neuroretina cells: an in vitro system to study crystallin aggregation process.

Transdifferentiated embryonic quail neuroretina cells synthesize in vitro crystallins (the lens-specific proteins) and form lentoid bodies (structures that mimic lens fiber cells) which also contain crystallins. A comparative study on the size of crystallins is reported in 7-day-old embryonic quail lenses, in 7-day-old embryonic quail transdifferentiated neuroretina cells (normal and MH2 transformed), and in isolated lentoid bodies. Analyses are performed using Superose FPLC in combination with SDS-PAGE and Western blot procedures. In quail lenses, an apparent 560-580-kDa alpha crystallin homopolymer is found and delta crystallin, the major avian lens protein, is detected as a 180-kDa tetramer. beta Crystallins, present in low amount within the 180-kDa peak, are a heterogeneous population composed of subunits of molecular weight identical to those found in chick lenses. In addition, an apparent 46-kDa monomeric delta crystallin is found. Normal and MH2-transformed neuroretina cultures produce an alpha crystallin polymer of lower molecular weight (450 kDa) and delta crystallin in a monomeric or dimeric form. The Western blot pattern of beta crystallins from MH2-transformed neuroretina cultures is strictly identical to that of quail lens beta crystallins. In particular, the beta B1 crystallin, which is specific to lens fiber cell differentiation, and the major beta 25-kDa crystallin are present. However, analysis of isolated lentoid bodies from normal transdifferentiated quail neuroretina cultures showed alpha and delta crystallins of comparable size to those found in lens extract, in particular the delta crystallin in tetrameric form. The lentoid body lens-like structure could favour the crystallin aggregation process.

Crystallin gene expression and lentoid body formation in quail embryo neuroretina cultures transformed by the oncogenic retrovirus Mill Hill 2 or Rous sarcoma virus.

The lens-specific proteins alpha and delta crystallins and lentoid bodies, structures that follow a differentiation pathway similar to that of the lens, regularly appear after 4 to 5 weeks in quail embryo neuroretina monolayer cultures. We have investigated the effects of the avian oncogenic retroviruses Mill Hill 2 and Rous sarcoma virus on this process. Quail embryo neuroretina cells transformed by Mill Hill 2 virus were established into permanent cultures that synthesized alpha and delta crystallins and contained stem cells for the production of lentoid bodies. In contrast, transformation with the Rous sarcoma virus mutant tsNY-68 blocked the appearance of mRNA crystallins, but cytoplasmic alpha and delta crystallin mRNA and alpha crystallin appeared 44 h after a shift to the nonpermissive temperature. However, delta crystallins and lentoid bodies were only present after 7 days. The crystallins of transformed quail neuroretina cultures were immunologically indistinguishable from those of quail lenses and of normal quail embryo neuroretina cultures.

State of differentiation of bovine epithelial lens cells in vitro. Relationship between the variation of the cell shape and the synthesis of crystallins.

Correlations between cell morphology and the expression of specific proteins (crystallins) have been investigated. Two different culture conditions have been chosen which keep bovine epithelial lens cells (BEL cells) in a monolayer of homogeneous epithelioid cells: (1) bovine retinal extract (EDGF) supplemented medium; (2) extracellular matrix (ECM) provided by corneal endothelial cells in standard medium has been compared to previous results obtained with BEL cells cultivated on plastic (Simonneau et al., 1983). Variations of the cell shape had no effect upon crystallin synthesis.

State of differentiation of bovine epithelial lens cells in vitro. Modulation of the synthesis and of the polymerization of specific proteins (crystallins) and non-specific proteins in relation to cell divisions.

Maintenance of the state of differentiation in serially cultured bovine epithelial lens cells has been investigated. The radioactive labelled soluble proteins were studied by gel filtration and gel electrophoresis. 1. In the lens epithelium on its capsule, preferential synthesis of alpha B2 vs alpha A2 crystallin subunits and synthesis of beta-crystallins (mainly beta Bp) were observed. 2. Epithelial lens cells cultured on plastic Petri dishes for up to 35 divisions still synthesized alpha B2 and beta Bp, but no longer alpha A2. Conversely, the same cells injected into nude mice synthesized alpha B and alpha A, but no beta-crystallin could be detected. 3. The ratio of non-crystallin proteins to crystallin polypeptides increased drastically with the number of cell divisions. Among these proteins, both Mr 45 000 and Mr 57 000 proteins are probably constituents of the water-soluble cytoskeletal proteins, respectively actin and vimentin. A Mr 17 000 polypeptide was observed and its relationship with a metabolic product of alpha-crystallin is proposed. 4. The polymerization process of crystallin polypeptides in these cells was studied and compared with crystallin aggregates found in the lens. Newly synthesized alpha crystallins were readily involved in high molecular aggregates. This process does not seem to require alpha A, since only alpha B was detected. Interestingly, non-crystallin-soluble proteins form the bulk of proteins found in high molecular weight (HMW) polymers. The time course of crystallin aggregate formation, in long-term culture cells, seems to be different for alpha- vs beta-polypeptides. These results allowed us to conclude that bovine epithelial lens cells in vitro, although they do not undergo terminal differentiation into fibers, are not dedifferentiated, since they still express specific features of the epithelium in situ.

DNA repair in lens cells during chick embryo development.

When chick lens epithelium is cultured in vitro, differentiation into lens fiber cells is accompanied by DNA degradation. This phenomenom of terminal differentiation was studied in the epithelium from embryos at the 6th and 11th days of development. DNA size and the ability of the cells to repair DNA damage induced by X-rays were analysed in alkaline sucrose gradients. In the 6-day epithelium a rapid degradation and complete lack of DNA repair were recorded. Similar observations have been made in previous studies on the 11-day sample, but here degradation is progressive and occurs after a lag of several days. In the younger epithelium, internal irradiation by [3H]thymidine also had a drastic effect resembling that caused by X-rays. In order to assess the process of differentiation in our experimental system the synthesis of delta- and alpha-crystallins was monitored. Stage-related modifications in the rates of synthesis were recorded. The results confirm that the DNA repair system is impaired during terminal differentiation. The differences observed between the two stages may reflect either a developmental modification in DNA repair mechanisms or a change in the relative proportions of differentiating cells. An hypothesis is proposed in support of the latter case.

Spontaneous transformation of bovine lens epithelial cells: kinetic analysis and differentiation in monolayers and in nude mice.

Bovine lens epithelial cells, in vivo, are known to perform two determined functions. First, they synthesize the lens capsule and subsequently, in the germinal region, they differentiate in fiber cells with massive production of crystallin proteins, inactivation and pyknosis of the nucleus. Bovine lens epithelial cells from adult origin can be cultured but so far no massive crystallin production has been demonstrated in vitro. We have studied the growth and differentiation of these cells and shown that in long term culture they acquire spontaneously many characteristics of transformation: unlimited growth potential, abnormal karyotype, multilayering. Viral particles were scarcely detected. However, they retain their epithelioid character and the ability to synthesize lens capsule material. Kinetic characteristics of those cells have been determined. When injected into nude mice, they actively proliferate and form tumors in which synthesis of alpha-crystallin can be demonstrated. These results show that in vitro transformation of lens epithelial cells does not affect their potential for terminal differentiation.