Characterization of paralogous uncx transcription factor encoding genes in zebrafish.

The paired-type homeodomain transcription factor Uncx is involved in multiple processes of embryogenesis in vertebrates. Reasoning that zebrafish genes uncx4.1 and uncx are orthologs of mouse Uncx, we studied their genomic environment and developmental expression. Evolutionary analyses indicate the zebrafish uncx genes as being paralogs deriving from teleost-specific whole-genome duplication. Whole-mount in situ mRNA hybridization of uncx transcripts in zebrafish embryos reveals novel expression domains, confirms those previously known, and suggests sub-functionalization of paralogs. Using genetic mutants and pharmacological inhibitors, we investigate the role of signaling pathways on the expression of zebrafish uncx genes in developing somites. In identifying putative functional role(s) of zebrafish uncx genes, we hypothesized that they encode transcription factors that coordinate growth and innervation of somitic muscles.

Characterization of paralogous uncx transcription factor encoding genes in zebrafish.

The paired-type homeodomain transcription factor Uncx is involved in multiple processes of embryogenesis in vertebrates. Reasoning that zebrafish genes uncx4.1 and uncx are orthologs of mouse Uncx, we studied their genomic environment and developmental expression. Evolutionary analyses indicate the zebrafish uncx genes as being paralogs deriving from teleost-specific whole-genome duplication. Whole-mount in situ mRNA hybridization of uncx transcripts in zebrafish embryos reveals novel expression domains, confirms those previously known, and suggests sub-functionalization of paralogs. Using genetic mutants and pharmacological inhibitors, we investigate the role of signaling pathways on the expression of zebrafish uncx genes in developing somites. In identifying putative functional role(s) of zebrafish uncx genes, we hypothesized that they encode transcription factors that coordinate growth and innervation of somitic muscles.

Temporal and spatial expression of insulin-like peptide (insl5a and insl5b) paralog genes during the embryogenesis of Danio rerio.

Relaxin (RLN) and insulin (INSL)-like peptides are member of the INSL/RLN superfamily, which are encoded by seven genes in humans and can activate the G-protein coupled receptor RXFP 1-4. These peptides evolved from a common ancestor, RLN3-like gene. Two rounds of whole genome duplication (WGD) in early vertebrate evolution, together with an additional WGD in the teleost lineage, caused an expansion of RLN genes set in the genome of Danio rerio. In particular, six RLN genes are present: a single copy of rln and insl3 genes, and two paralogs for the rln3 gene (rln3a and rln3b), and the insl5 gene (insl5a and insl5b). We have already reported the presence of rln3a and rln3b genes in the developing zebrafish brain, as well as the expression of rln gene in the developing zebrafish brain and extraneural territories, such as thyroid gland and pancreas. Here, we report for the first time the expression of the two parologs genes for insl5, insl5a, and insl5b in D. rerio embryonic development. The corresponding transcripts of both the paralogs are present in all embryonic stages analyzed by RT-qPCR. In situ hybridization analyses showed a restricted signal in intestinal cells and the pancreatic region at 72 hpf for insl5a, while at 96 hpf both genes are expressed in specific intestinal cells. Furthermore, in adult zebrafish intestine tissue, in situ hybridation experiments showed that insl5a transcript is specifically localized in the goblet cells, while insl5b transcript is in enteroendocrine cells. These data revealed a high degree of gene expression pattern conservation for such genes in vertebrate evolution.

Expression pattern of zebrafish rxfp2 homologue genes during embryonic development.

RXFP2 is one of the 4 receptors for relaxin insulin-like peptides, in particular it binds with high affinity the INSL3 peptide. INSL3/RXFP2 pair is essential for testicular descent during placental mammalian development. The evolutionary history of this ligand/receptor pair has received much attention, since its function in vertebrate species lacking testicular descent, such as the fishes, remains elusive. Herein, we analyzed the expression pattern of three rxfp2 homologue genes in zebrafish embryonic development. For all the three rxfp2 genes (rxfp2a, rxfp2b, and rxfp2-like) we showed the presence of maternally derived transcripts. Later in the development, rxfp2a is only expressed at larval stage, whereas rxfp2b is expressed in all the analyzed stage with highest level in the larvae. The rxfp2-like gene is expressed in all the analyzed stage with a transcript level that increased starting at early pharyngula stage. The spatial localization analysis of rxfp2-like gene showed that it is expressed in many cell clusters in the developing brain. In addition, other rxfp2-like-expressing cells were identified in the retina and oral epithelium. This analysis provides new insights to elucidate the evolution of rxfp2 genes in vertebrate lineage and lays the foundations to study their role in vertebrate embryonic development.

Expression analysis of five zebrafish RXFP3 homologues reveals evolutionary conservation of gene expression pattern.

Relaxin peptides exert different functions in reproduction and neuroendocrine processes via interaction with two evolutionarily unrelated groups of receptors: RXFP1 and RXFP2 on one hand, RXFP3 and RXFP4 on the other hand. Evolution of receptor genes after splitting of tetrapods and teleost lineage led to a different retention rate between mammals and fish, with the latter having more gene copies compared to the former. In order to improve our knowledge on the evolution of the relaxin ligands/receptors system and have insights on their function in early stages of life, in the present paper we analyzed the expression pattern of five zebrafish RXFP3 homologue genes during embryonic development. In our analysis, we show that only two of the five genes are expressed during embryogenesis and that their transcripts are present in all the developmental stages. Spatial localization analysis of these transcripts revealed that the gene expression is restricted in specific territories starting from early pharyngula stage. Both genes are expressed in the brain but in different cell clusters and in extra-neural territories, one gene in the interrenal gland and the other in the pancreas. These two genes share expression territories with the homologue mammalian counterpart, highlighting a general conservation of gene expression regulatory processes and their putative function during evolution that are established early in vertebrate embryogenesis.

Developmental expression pattern of two zebrafish rxfp3 paralogue genes.

In mammals, the RXFP3 is the cognate receptor of the relaxin-3 peptide (RLN3). In teleosts, many different orthologue genes for RXFP3 are present. In particular, two paralogue genes, rxfp3-2a and rxfp3-2b, likely encode the receptors for the Rln3a peptide. The transcription of these two rxfp3 genes is differentially regulated early during zebrafish embryogenesis. Indeed, reverse transcription-polymerase chain reaction analyses show that the rxfp3-2b transcript is always present during embryo development, while the rxfp3-2a transcript is detectable only at larval stage. By in situ hybridization experiments on embryos and larvae, the rxfp3-2b transcript was revealed in the brain and in the retinal ganglion cell layer and thymus. Particularly in the brain, many territories are involved in the rxfp3-2b expression, among them the optic tectum, thalamus, preoptic area, different nerve nuclei, habenula and pineal gland. The RXFP3 spatiotemporal expression pattern appears to be conserved between Danio rerio and mammals, as also previously showed for the corresponding ligand, the RLN3. Interestingly, the brain areas expressing the rxfp3-2b receptor gene are involved in the visual system, emotional behaviors and circadian rhythm and could be functionally related to the neurotransmitter Rln3a-expressing territories.

Characterization, cDNA cloning and expression pattern of relaxin gene during embryogenesis of Danio rerio.

We report the identification, the cDNA cloning, the temporal and spatial expression pattern analysis of the rln gene in the zebrafish Danio rerio. The deduced Rln B and A domains show different evolutionary conservation. Rln B domain shows higher similarity when compared to zebrafish and human RLN3 B domain than human RLN1 and RLN2 B domain. Differently, the zebrafish Rln A domain shows relatively low amino acid sequence similarity when compared with the same sequences. The rln gene is transcribed both during embryogenesis and in adult organism, where higher transcript level has been particularly evidenced in the brain. Moreover, we provide the first description of rln spatial expression pattern during embryonic development. In particular, we show restricted transcript localization starting at the pharyngula stage in olfactory placode, branchial arch region, and in a cell cluster near to otic vesicle. In larval stage, new transcription territories have been detected in both neural and non-neural regions. In particular, in the brain, rln expression has been revealed in telencephalic region around anterior commissure, in the preoptic area, and in restricted rombencephalic cell clusters. Expression of rln gene in extra-neural territories has been detected in the pancreatic and thyroid gland regions. Danio rerio rln expression pattern analysis reveals shared features with the mammalian RLN gene, particularly in the brain, where it might have a role in the neurophysiological processes. In addition, expression in the thyroid and pancreas region suggests a function as a paracrine and endocrine hormone.

Characterization and developmental expression pattern of the relaxin receptor rxfp1 gene in zebrafish.

We report the gene characterization, the cDNA cloning and the temporal and spatial expression pattern of the relaxin receptor rxfp1 gene in the zebrafish Danio rerio. The zebrafish rxfp1 gene has the same syntenic genomic organization, and a similar exon-intron structure to the homologue human gene. Furthermore, the deduced Rxfp1 protein sequence shows a high degree of amino acid similarity when compared with the human protein and the conservation of all amino acid identity necessary for the binding with relaxin. Our results show that rxfp1 gene is active either during embryogenesis or in the adult organism, showing a wide expression pattern. Moreover, we provide the first description of rxfp1 spatial expression pattern during embryo development, showing that the transcript is already present at the early developmental stage and is distributed in all of the embryonic cells until somitogenesis. Starting at the pharyngula stage the gene expression becomes mainly restricted in the brain territories. In fact, at the larval stage, the transcript is detectable in the epiphysis, postoptic region, posterior tuberculum, hypothalamus, optic tectum, tegmentum/pons, medulla and also in the structure of a peripheral nervous system, the terminal nerve. The rxfp1 expression pattern in Danio rerio embryos is very similar to that reported in the adult mammalian brain, suggesting a pivotal role of this receptor in the neurophysiology processes already at very early developmental stages.

Duplicated zebrafish relaxin-3 gene shows a different expression pattern from that of the co-orthologue gene.

Relaxin-3 (Rln3) is thought to function as a neurotransmitter mainly produced in the mammalian nucleus incertus and is involved in different neural processes; among them, the stress response and food intake. Here, we report the expression pattern of the duplicated zebrafish rln3b gene and compare it to the previously analyszd spatial expression pattern of the rln3a gene. Both genes, during the embryogenesis and in the adult fish, are active and show relevant differences in their expression patterns. rln3b is diffusely expressed in the brain until the pharyngula period, when, at 48 h postfertilization (hpf), the expression becomes restricted to the periaqueductal gray, where it persists also at later developmental stages. No expression was observed in the nucleus incertus cells that express the rln3a gene from 72 hpf. In the adult, both genes are expressed in brain, but only rln3b transcript is revealed in testis at the similar expression level, whereas in the other analyzed tissues the transcript levels are lower or absent. Both the putative mature protein sequences are highly conserved, this feature and their differential expression patterns might indicate a sub-functionalization during evolution with the consequent retention of the two paralogues genes.