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 szed. 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.

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.

Myelin-associated axon growth inhibitors such as Nogo-A/RTN4-A impair axon regeneration in the adult mammalian central nervous system (CNS). Here, we describe the cloning and expression of two independent Xenopus laevis rtn4 orthologs. As in mammals, alternative transcripts are generated both through differential splicing and promoter usage, giving rise to Xenopus nogo-A, -B, -C and to a new isoform, nogo-N/rtn4-N. Xenopus is therefore the ‘lowest’ vertebrate where Nogo-A was identified. Xenopus Nogo-A/RTN4-A is predominantly expressed in the nervous system, whereas the other isoforms mainly occur in nonneuronal tissues. Nogo-A/RTN4-A specific antisera detect the protein in myelinated fiber tracts of the spinal cord, hindbrain, optic nerve, tectum opticum and in isolated oligodendrocytes. In addition, subpopulations of CNS neurons are Nogo-A/RTN4-A positive. This expression pattern is consistent with that observed for rat Nogo-A and suggests similar functions. Nogo-A in Xenopus myelin might therefore contribute to the failure of spinal cord regeneration in frogs-a feature that may have evolved during the transition from fish to land vertebrates.

The Nogo-66 receptor NgR has been implicated in the mediation of inhibitory effects of central nervous system (CNS) myelin on axon growth in the adult mammalian CNS. NgR binds to several myelin-associated ligands (Nogo-66, myelin associated glycoprotein, and oligodendrocyte-myelin glycoprotein), which, among other inhibitory proteins, impair axonal regeneration in the CNS of adult mammals. In contrast to mammals, severed axons readily regenerate in the fish CNS. Nevertheless, fish axons are repelled by mammalian oligodendrocytes in vitro. Therefore, the identification of fish NgR homologs is a crucial step towards understanding NgR functions in vertebrate systems competent of CNS regeneration. Here, we report the discovery of four zebrafish (Danio rerio) and five fugu (Takifugu rubripes) NgR homologs. Synteny between fish and human, comparable intron-exon structures, and phylogenetic analyses provide convincing evidence that the true fish orthologs were identified. The topology of the phylogenetic trees shows that the extra fish genes were produced by duplication events that occurred in ray-finned fishes before the divergence of the zebrafish and pufferfish lineages. Expression of zebrafish NgR homologs was detected relatively early in development and prominently in the adult brain, suggesting functions in axon growth, guidance, or plasticity.

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.

In many organisms, the primordial germ cells have to migrate from the position where they are specified towards the developing gonad where they generate gametes. Extensive studies of the migration of primordial germ cells in Drosophila, mouse, chick and Xenopus have identified somatic tissues important for this process and demonstrated a role for specific molecules in directing the cells towards their target. In zebrafish, a unique situation is found in that the primordial germ cells, as marked by expression of vasa mRNA, are specified in random positions relative to the future embryonic axis. Hence, the migrating cells have to navigate towards their destination from various starting positions that differ among individual embryos. Here, we present a detailed description of the migration of the primordial germ cells during the first 24 hours of wild-type zebrafish embryonic development. We define six distinct steps of migration bringing the primordial germ cells from their random positions before gastrulation to form two cell clusters on either side of the midline by the end of the first day of development. To obtain information on the origin of the positional cues provided to the germ cells by somatic tissues during their migration, we analyzed the migration pattern in mutants, including spadetail, swirl, chordino, floating head, cloche, knypek and no isthmus. In mutants with defects in axial structures, paraxial mesoderm or dorsoventral patterning, we find that certain steps of the migration process are specifically affected. We show that the paraxial mesoderm is important for providing proper anteroposterior information to the migrating primordial germ cells and that these cells can respond to changes in the global dorsoventral coordinates. In certain mutants, we observe accumulation of ectopic cells in different regions of the embryo. These ectopic cells can retain both morphological and molecular characteristics of primordial germ cells, suggesting that, in zebrafish at the early stages tested, the vasa-expressing cells are committed to the germ cell lineage.