A Foxf1-Wnt-Nr2f1 cascade promotes atrial cardiomyocyte differentiation in zebrafish.

Nr2f transcription factors (TFs) are conserved regulators of vertebrate atrial cardiomyocyte (AC) differentiation. However, little is known about the mechanisms directing Nr2f expression in ACs. Here, we identified a conserved enhancer 3' to the nr2f1a locus, which we call 3'reg1-nr2f1a (3'reg1), that can promote Nr2f1a expression in ACs. Sequence analysis of the enhancer identified putative Lef/Tcf and Foxf TF binding sites. Mutation of the Lef/Tcf sites within the 3'reg1 reporter, knockdown of Tcf7l1a, and manipulation of canonical Wnt signaling support that Tcf7l1a is derepressed via Wnt signaling to activate the transgenic enhancer and promote AC differentiation. Similarly, mutation of the Foxf binding sites in the 3'reg1 reporter, coupled with gain- and loss-of-function analysis supported that Foxf1 promotes expression of the enhancer and AC differentiation. Functionally, we find that Wnt signaling acts downstream of Foxf1 to promote expression of the 3'reg1 reporter within ACs and, importantly, both Foxf1 and Wnt signaling require Nr2f1a to promote a surplus of differentiated ACs. CRISPR-mediated deletion of the endogenous 3'reg1 abrogates the ability of Foxf1 and Wnt signaling to produce surplus ACs in zebrafish embryos. Together, our data support that downstream members of a conserved regulatory network involving Wnt signaling and Foxf1 function on a nr2f1a enhancer to promote AC differentiation in the zebrafish heart.

Gene expression in notochord and nuclei pulposi: a study of gene families across the chordate phylum.

The transition from notochord to vertebral column is a crucial milestone in chordate evolution and in prenatal development of all vertebrates. As ossification of the vertebral bodies proceeds, involutions of residual notochord cells into the intervertebral discs form the nuclei pulposi, shock-absorbing structures that confer flexibility to the spine. Numerous studies have outlined the developmental and evolutionary relationship between notochord and nuclei pulposi. However, the knowledge of the similarities and differences in the genetic repertoires of these two structures remains limited, also because comparative studies of notochord and nuclei pulposi across chordates are complicated by the gene/genome duplication events that led to extant vertebrates. Here we show the results of a pilot study aimed at bridging the information on these two structures. We have followed in different vertebrates the evolutionary trajectory of notochord genes identified in the invertebrate chordate Ciona, and we have evaluated the extent of conservation of their expression in notochord cells. Our results have uncovered evolutionarily conserved markers of both notochord development and aging/degeneration of the nuclei pulposi.

Origin and evolutionary landscape of Nr2f transcription factors across Metazoa.

BACKGROUND: Nuclear Receptor Subfamily 2 Group F (Nr2f) orphan nuclear hormone transcription factors (TFs) are fundamental regulators of many developmental processes in invertebrates and vertebrates. Despite the importance of these TFs throughout metazoan development, previous work has not clearly outlined their evolutionary history. RESULTS: We integrated molecular phylogeny with comparisons of intron/exon structure, domain architecture, and syntenic conservation to define critical evolutionary events that distinguish the Nr2f gene family in Metazoa. Our data indicate that a single ancestral eumetazoan Nr2f gene predated six main Bilateria subfamilies, which include single Nr2f homologs, here referred to as Nr2f1/2/5/6, that are present in invertebrate protostomes and deuterostomes, Nr2f1/2 homologs in agnathans, and Nr2f1, Nr2f2, Nr2f5, and Nr2f6 orthologs that are found in gnathostomes. Four cnidarian Nr2f1/2/5/6 and three agnathan Nr2f1/2 members are each due to independent expansions, while the vertebrate Nr2f1/Nr2f2 and Nr2f5/Nr2f6 members each form paralogous groups that arose from the established series of whole-genome duplications (WGDs). Nr2f6 members are the most divergent Nr2f subfamily in gnathostomes. Interestingly, in contrast to the other gnathostome Nr2f subfamilies, Nr2f5 has been independently lost in numerous vertebrate lineages. Furthermore, our analysis shows there are differential expansions and losses of Nr2f genes in teleosts following their additional rounds of WGDs. CONCLUSION: Overall, our analysis of Nr2f gene evolution helps to reveal the origins and previously unrecognized relationships of this ancient TF family, which may allow for greater insights into the conservation of Nr2f functions that shape Metazoan body plans.

The Cis-Regulatory Code for Kelch-like 21/30 Specific Expression in Ciona robusta Sensory Organs.

The tunicate Ciona robusta is an emerging model system to study the evolution of the nervous system. Due to their small embryos and compact genomes, tunicates, like Ciona robusta, have great potential to comprehend genetic circuitry underlying cell specific gene repertoire, among different neuronal cells. Their simple larvae possess a sensory vesicle comprising two pigmented sensory organs, the ocellus and the otolith. We focused here on Klhl21/30, a gene belonging to Kelch family, that, in Ciona robusta, starts to be expressed in pigmented cell precursors, becoming specifically maintained in the otolith precursor during embryogenesis. Evolutionary analyses demonstrated the conservation of Klhl21/30 in all the chordates. Cis-regulatory analyses and CRISPR/Cas9 mutagenesis of potential upstream factors, revealed that Klhl21/30 expression is controlled by the combined action of three transcription factors, Mitf, Dmrt, and Msx, which are downstream of FGF signaling. The central role of Mitf is consistent with its function as a fundamental regulator of vertebrate pigment cell development. Moreover, our results unraveled a new function for Dmrt and Msx as transcriptional co-activators in the context of the Ciona otolith.

Rimbp, a New Marker for the Nervous System of the Tunicate Ciona robusta.

Establishment of presynaptic mechanisms by proteins that regulate neurotransmitter release in the presynaptic active zone is considered a fundamental step in animal evolution. Rab3 interacting molecule-binding proteins (Rimbps) are crucial components of the presynaptic active zone and key players in calcium homeostasis. Although Rimbp involvement in these dynamics has been described in distantly related models such as fly and human, the role of this family in most invertebrates remains obscure. To fill this gap, we defined the evolutionary history of Rimbp family in animals, from sponges to mammals. We report, for the first time, the expression of the two isoforms of the unique Rimbp family member in Ciona robusta in distinct domains of the larval nervous system. We identify intronic enhancers that are able to drive expression in different nervous system territories partially corresponding to Rimbp endogenous expression. The analysis of gene expression patterns and the identification of regulatory elements of Rimbp will positively impact our understanding of this family of genes in the context of Ciona embryogenesis.

Transphyletic conservation of nitric oxide synthase regulation in cephalochordates and tunicates.

Nitric oxide synthase is ubiquitously present in metazoans and is involved in a wide range of biological processes. Three distinct Nos genes have been so far identified in vertebrates exhibiting a complex expression pattern and transcriptional regulation. Nevertheless, although independent events of Nos duplication have been observed in several taxa, only few studies described the regulatory mechanisms responsible for their activation in non-vertebrate animals. To shed light on the mechanisms underlying neuronal-type Nos expression, we focused on two non-vertebrate chordates: the cephalochordate Branchiostoma lanceolatum and the tunicate Ciona robusta. Here, throughout transphyletic and transgenic approaches, we identified genomic regions in both species acting as Nos functional enhancers during development. In vivo analyses of Nos genomic fragments revealed their ability to recapitulate the endogenous expression territories. Therefore, our results suggest the existence of evolutionary conserved mechanisms responsible for neuronal-type Nos regulation in non-vertebrate chordates. In conclusion, this study paves the way for future characterization of conserved transcriptional logic underlying the expression of neuronal-type Nos genes in chordates.

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.

The evolutionary landscape of the Rab family in chordates.

Intracellular traffic amongst organelles represents a key feature for eukaryotes and is orchestrated principally by members of Rab family, the largest within Ras superfamily. Given that variations in Rab repertoire have been fundamental in animal diversification, we provided the most exhaustive survey regarding the Rab toolkit of chordates. Our findings reveal the existence of 42 metazoan conserved subfamilies exhibiting a univocal intron/exon structure preserved from cnidarians to vertebrates. Since the current view does not capture the Rab complexity, we propose a new Rab family classification in three distinct monophyletic clades. The Rab complement of chordates shows a dramatic diversification due to genome duplications and independent gene duplications and losses with sharp differences amongst cephalochordates, tunicates and gnathostome vertebrates. Strikingly, the analysis of the domain architecture of this family highlighted the existence of chimeric calcium-binding Rabs, which are animal novelties characterized by a complex evolutionary history in gnathostomes and whose role in cellular metabolism is obscure. This work provides novel insights in the knowledge of Rab family: our hypothesis is that chordates represent a hotspot of Rab variability, with many events of gene gains and losses impacting intracellular traffic capabilities. Our results help to elucidate the role of Rab members in the transport amongst endomembranes and shed light on intracellular traffic routes in vertebrates. Then, since the predominant role of Rabs in the molecular communication between different cellular districts, this study paves to way to comprehend inherited or acquired human disorders provoked by dysfunctions in Rab genes.

Transcriptional regulation of Rab32/38, a specific marker of pigment cell formation in Ciona robusta.

Through a myriad of pigments stored in different cells, animal pigmentation represents a crucial process to face disparate environmental and ecological challenges. In vertebrates, the small GTPase Rab32 and Rab38 have a conserved role in the transport of key melanogenic enzymes, as tyrosinase (tyr) and tyrosinase-related protein (tyrp), to the melanosomes in formation. We provide a survey on Rab32/38 evolution and its regulatory logics during pigment cell formation in Ciona robusta. Our phylogeny supports the existence of a single Rab32/38 gene in tunicates, which is probably the unique transporter for tyrosinase family members in this clade. Different deletions allow us to identify the minimal cis-regulatory element able to recapitulate the endogenous gene expression during pigment cell development in C. robusta. In this conserved region, we identified two putative binding sites for the transcription factor Mitf, which is known for its role as regulator of pigmentation in vertebrates. Mutational analysis revealed that both Mitf binding sites are essential for the activity of this regulatory region and we demonstrated that Mitf misexpression is able to induce ectopic activation of the Rab32/38 regulatory region in vivo. Our results strongly indicate that Mitf is involved in the regulation of Rab32/38 activity during Ciona pigment cell development.

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.

Ran GTPase, an eukaryotic gene novelty, is involved in amphioxus mitosis.

Ran (ras-related nuclear protein) is a small GTPase belonging to the RAS superfamily that is specialized in nuclear trafficking. Through different accessory proteins, Ran plays key roles in several processes including nuclear import-export, mitotic progression and spindle assembly. Consequently, Ran dysfunction has been linked to several human pathologies. This work illustrates the high degree of amino acid conservation of Ran orthologues across evolution, reflected in its conserved role in nuclear trafficking. Moreover, we studied the evolutionary scenario of the pre-metazoan genetic linkage between Ran and Stx, and we hypothesized that chromosomal proximity of these two genes across metazoans could be related to a regulatory logic or a functional linkage. We studied, for the first time, Ran expression during amphioxus development and reported its presence in the neural vesicle, mouth, gill slits and gut corresponding to body regions involved in active cell division.

A comprehensive analysis of neurotrophins and neurotrophin tyrosine kinase receptors expression during development of zebrafish.

Neurotrophins (NTF) are a family of secreted nerve growth factors with affinity for tyrosine kinase (Ntrk) and p75 receptors. To fully understand the variety of developmental roles played by NTFs, it is critical to know when and where genes encoding individual ligands and receptors are transcribed. Identification of ntf and ntrk transcripts in zebrafish development remains to be fully characterized for further uncovering the potential function(s) of the NTF signal transduction pathway. Here, we conducted a systematic analysis of the expression profiles of four ntf and five ntrk genes during zebrafish development using whole-mount in situ hybridization. Our study unveils new expression domains in the developing embryo, confirms those previously known, and shows that ntf and ntrk genes have different degrees of cell- and tissue-type specificity. The unique and overlapping expression patterns here depicted indicate the coordination of the redundant and divergent functions of NTFs and represent valuable tools for deciphering the molecular pathways involved in the specification and function of embryonic cell types.

Evolutionary loss of melanogenesis in the tunicate Molgula occulta.

BACKGROUND: Analyzing close species with diverse developmental modes is instrumental for investigating the evolutionary significance of physiological, anatomical and behavioral features at a molecular level. Many examples of trait loss are known in metazoan populations living in dark environments. Tunicates are the closest living relatives of vertebrates and typically present a lifecycle with distinct motile larval and sessile adult stages. The nervous system of the motile larva contains melanized cells associated with geotactic and light-sensing organs. It has been suggested that these are homologous to vertebrate neural crest-derived melanocytes. Probably due to ecological adaptation to distinct habitats, several species of tunicates in the Molgulidae family have tailless (anural) larvae that fail to develop sensory organ-associated melanocytes. Here we studied the evolution of Tyrosinase family genes, indispensible for melanogenesis, in the anural, unpigmented Molgula occulta and in the tailed, pigmented Molgula oculata by using phylogenetic, developmental and molecular approaches. RESULTS: We performed an evolutionary reconstruction of the tunicate Tyrosinase gene family: in particular, we found that M. oculata possesses genes predicted to encode one Tyrosinase (Tyr) and three Tyrosinase-related proteins (Tyrps) while M. occulta has only Tyr and Tyrp.a pseudogenes that are not likely to encode functional proteins. Analysis of Tyr sequences from various M. occulta individuals indicates that different alleles independently acquired frameshifting short indels and/or larger mobile genetic element insertions, resulting in pseudogenization of the Tyr locus. In M. oculata, Tyr is expressed in presumptive pigment cell precursors as in the model tunicate Ciona robusta. Furthermore, a M. oculata Tyr reporter gene construct was active in the pigment cell precursors of C. robusta embryos, hinting at conservation of the regulatory network underlying Tyr expression in tunicates. In contrast, we did not observe any expression of the Tyr pseudogene in M. occulta embryos. Similarly, M. occulta Tyr allele expression was not rescued in pigmented interspecific M. occulta × M. oculata hybrid embryos, suggesting deleterious mutations also to its cis-regulatory sequences. However, in situ hybridization for transcripts from the M. occulta Tyrp.a pseudogene revealed its expression in vestigial pigment cell precursors in this species. CONCLUSIONS: We reveal a complex evolutionary history of the melanogenesis pathway in tunicates, characterized by distinct gene duplication and loss events. Our expression and molecular data support a tight correlation between pseudogenization of Tyrosinase family members and the absence of pigmentation in the immotile larvae of M. occulta. These results suggest that relaxation of purifying selection has resulted in the loss of sensory organ-associated melanocytes and core genes in the melanogenesis biosynthetic pathway in M. occulta.

Expression of caspase 3 in ovarian follicle cells of the lizard Podarcis sicula.

In this study, our aim was to determine whether caspase 3 plays a role, during previtellogenesis, in the ovarian follicular epithelium of the lizard Podarcis sicula. We investigated the presence and localization of proform and active caspase 3 by enzyme assay, Western blotting and immunocytochemistry. In parallel, a fragment of caspase 3 was cloned for the first time in this species, sequenced and used for in situ hybridization to localize messengers and analysed by a phylogenetic survey to shed light on its homology with reptilian caspases. Results demonstrated that: (1) the follicle cells expressed a caspase of the 3/7 group and the mRNA for caspase 3 was transcribed in the stem phase and was completely translated during cell differentiation; (2) the proform protein was stored during the differentiated (nurse) stage and activated at the end of previtellogenesis provoking the degeneration of cells; (3) the predicted protein sequence, although partial, had a strong similarity with the known reptilian caspases 3. The epithelial cells of the ovarian follicle, therefore, do not employ caspase 3 during the nurse stage but, instead, prepare for apoptosis long before the process actually begins. The relevance of this strategy is discussed.

Rab32 and Rab38 genes in chordate pigmentation: an evolutionary perspective.

BACKGROUND: The regulation of cellular membrane trafficking in all eukaryotes is a very complex mechanism, mostly regulated by the Rab family proteins. Among all membrane-enclosed organelles, melanosomes are the cellular site for synthesis, storage and transport of melanin granules, making them an excellent model for studies on organelle biogenesis and motility. Specific Rab proteins, as Rab32 and Rab38, have been shown to play a key role in melanosome biogenesis. We analysed the Rab32 and Rab38 genes in the teleost zebrafish and in the cephalochordate amphioxus, gaining insight on their evolutionary history following gene and genome duplications. RESULTS: We studied the molecular evolution of Rab supergroup III in deuterostomes by phylogenetic reconstruction, intron and synteny conservation. We discovered a novel amino acid stretch, named FALK, shared by three related classes belonging to Rab supergroup III: Rab7L1, Rab32LO and Rab32/Rab38. Among these, we demonstrated that the Rab32LO class, already present in the last common eukaryotic ancestor, was lost in urochordates and vertebrates. Synteny shows that one zebrafish gene, Rab38a, which is expressed in pigmented cells, retained the linkage with tyrosinase, a protein essential for pigmentation. Moreover, the chromosomal linkage of Rab32 or Rab38 with a member of the glutamate receptor metabotropic (Grm) family has been retained in all analysed gnathostomes, suggesting a conserved microsynteny in the vertebrate ancestor. Expression patterns of Rab32 and Rab38 genes in zebrafish, and Rab32/38 in amphioxus, indicate their involvement in development of pigmented cells and notochord. CONCLUSIONS: Phylogenetic, intron conservation and synteny analyses point towards an evolutionary scenario based on a duplication of a single invertebrate Rab32/38 gene giving rise to vertebrate Rab32 and Rab38. The expression patterns of Rab38 paralogues highlight sub-functionalization event. Finally, the discovery of a chromosomal linkage between the Rab32 or Rab38 gene with a Grm opens new perspectives on possible conserved bystander gene regulation across the vertebrate evolution.