Reticulon (RTN) genes code for a family of proteins relatively recently described in higher vertebrates. The four known mammalian paralogues (RTN1, -2, -3, and -4/Nogo) have homologous carboxyl termini with two characteristic large hydrophobic regions. Except for RTN4-A/Nogo-A, thought to be an inhibitor for neurite outgrowth, restricting the regenerative capabilities of the mammalian CNS after injury, the functions of other family members are largely unknown. The overall occurrence of RTNs in different phyla and the evolution of the RTN gene family have hitherto not been analyzed. Here we expound data showing that the RTN family has arisen during early eukaryotic evolution potentially concerted to the establishment of the endomembrane system. Over 250 reticulon-like (RTNL) genes were identified in deeply diverging eukaryotes, fungi, plants, and animals. A systematic nomenclature for all identified family members is introduced. The analysis of exon-intron arrangements and of protein homologies allowed us to isolate key steps in the history of these genes. Our data corroborate the hypothesis that present RTNs evolved from an intron-rich reticulon ancestor mainly by the loss of different introns in diverse phyla. We also present evidence that the exceptionally large RTN4-A-specific exon 3, which harbors a potent neurite growth inhibitory region, may have arisen de novo approximately 350 MYA during transition to land vertebrates. These data emphasize on the one hand the universal role of reticulons in the eukaryotic system and on the other hand the acquisition of putative new functions through acquirement of novel amino-terminal exons.

The hyaluronic acid binding region was prepared by clostripain digestion of chondroitin sulfate proteoglycan isolated from the Swarm rat chondrosarcoma, and biotinylated in the presence of associated hyaluronic acid and link protein. After removal of hyaluronic acid by gel filtration in 4 M guanidine HCl, the biotinylated binding region-link protein complex was used as a specific histochemical probe in conjunction with avidin-peroxidase. Its utility was initially evaluated by comparison with Alcian blue staining of the axial region of 2 to 5 day chick embryos, where staining was seen in the dorsolateral area between the neural tube and the ectoderm, in the perichordal mesenchyme, and in developing limb buds. Light and electron microscopic studies of early postnatal rat cerebellum indicate that hyaluronic acid is primarily localized in the extracellular space of immature brain. Staining specificity was demonstrated by the ability of hyaluronic acid oligosaccharides of appropriate size to block the staining reaction, and by the absence of staining after treatment of tissue sections with protease-free Streptomyces hyaluronidase, which degrades only this glycosaminoglycan.

Reticulons (RTNs) are a family of evolutionary conserved proteins with four RTN paralogs (RTN1, RTN2, RTN3, and RTN4) present in land vertebrates. While the exact functions of RTN1 to RTN3 are unknown, mammalian RTN4-A/Nogo-A was shown to inhibit the regeneration of severed axons in the mammalian central nervous system (CNS). This inhibitory function is exerted via two distinct regions, one within the Nogo-A-specific N-terminus and the other in the conserved reticulon homology domain (RHD). In contrast to mammals, fish are capable of CNS axon regeneration. We performed detailed analyses of the fish rtn gene family to determine whether this regeneration ability correlates with the absence of the neurite growth inhibitory protein Nogo-A. A total of 7 rtn genes were identified in zebrafish, 6 in pufferfish, and 30 in eight additional fish species. Phylogenetic and syntenic relationships indicate that the identified fish rtn genes are orthologs of mammalian RTN1, RTN2, RTN3, and RTN4 and that several paralogous fish genes (e.g., rtn4 and rtn6) resulted from genome duplication events early in actinopterygian evolution. Accordingly, sequences homologous to the conserved RTN4/Nogo RHD are present in two fish genes, rtn4 and rtn6. However, sequences comparable to the first approximately 1,000 amino acids of mammalian Nogo-A including a major neurite growth inhibitory region are absent in zebrafish. This result is in accordance with functional data showing that axon growth inhibitory molecules are less prominent in fish oligodendrocytes and CNS myelin compared to mammals.

The widespread success of clinical implantology stems from bone's ability to form rigid, load-bearing connections to titanium and certain bioactive coatings. Adhesive biomolecules in the extracellular matrix are presumably responsible for much of the strength and stability of these junctures. Histochemical and spectroscopic analyses of retrievals have been supplemented by studies of osteoblastic cells cultured on implant materials and of the adsorption of biomolecules to titanium powder. These data have often been interpreted to suggest that proteoglycans permeate a thin, collagen-free zone at the most intimate contact points with implant surfaces. This conclusion has important implications for the development of surface modifications to enhance osseointegration. The evidence for proteoglycans at the interface, however, is somewhat less than compelling due to the lack of specificity of certain histochemical techniques and to possible sectioning artifacts. With this caveat in mind, we have devised a working model to explain certain observations of implant interfaces in light of the known physical and biological properties of bone proteoglycans. This model proposes that titanium surfaces accelerate osseointegration by causing the rapid degradation of a hyaluronan meshwork formed as part of the wound-healing response. It further suggests that the adhesive strength of the thin, collagen-free zone is provided by a bilayer of decorin proteoglycans held in tight association by their overlapping glycosaminoglycan chains.

The Cntn1 (Contactin/F3/F11) cell adhesion molecule is involved in axon growth and guidance, fasciculation, synapse formation, and myelination in birds and mammals. We identified Cntn1 genes in goldfish, zebrafish, and fugu, and provide evidence for a fish-specific duplication leading to Cntn1a and Cntn1b. Our analyses suggest a subfunctionalization for the Cntn1 paralogs in zebrafish compared to other vertebrates which have a single Cntn1 gene. Similar to Cntn1a, Cntn1b transcripts are found in subsets of sensory and motor neurons. However, Cntn1b is detected later and more restricted than Cntn1a. This spatio-temporal expression pattern of the two zebrafish Cntn1 paralogs suggests functions related to those of mammalian Cntn1. In adult goldfish, Cntn1b is expressed in oligodendrocytes and is upregulated in retinal ganglion cells after optic nerve transection, which is consistent with an additional role during regeneration.