RESUMEN

Cystatins are well known as a vast superfamily of functional proteins participated in the reversible competitive inhibition of cysteine proteases. Currently, increasing evidences point to the extensive phylogenetic diversity and crucial immune roles of type-2 cystatins in the vertebrate species. However, no information is available regarding the homologue in cephalochordate amphioxus, the representative of most basal living chordates, whose immune regulation are still ambiguous. Here, we clearly identified the presence of type-2 cystatin gene in amphioxus Branchiostoma japonicum, termed Bjcystatin-2, which was structurally characterized by typical wedge-shaped cystatin feature. Evolutionary analyses revealed that Bjcystatin-2 is the putative ancestral type-2 cystatin for chordates, with gene diversity emerging through duplication events. The expression of Bjcystatin-2 showed tissue-specific profile and was inducible upon invasive pathogens. Significantly, the recombinant Bjcystatin-2 exhibited not merely cathepsin L inhibitory activity, but also the ability to bind with bacteria and their characteristic molecules. Furthermore, Bjcystatin-2 also showed the capacity to enhance the macrophage-driven bacterial phagocytosis and to attenuate the generation of pro-inflammatory cytokines within macrophages. In summary, these findings demonstrate that Bjcystatin-2 exhibits dual role acting as both a protease inhibitor and an immunoactive molecule, greatly enriching our understanding of immune defense mechanisms of type-2 cystatin within the amphioxus.

Asunto(s)

RESUMEN

The Pax6 gene is essential for eye and brain development across various animal species. Here, we investigate the function of Pax6 in the development of the anterior central nervous system (CNS) of the invertebrate chordate amphioxus using CRISPR/Cas9-induced genome editing. Specifically, we examined Pax6 mutants featuring a 6 bp deletion encompassing two invariant amino acids in the conserved paired domain, hypothesized to impair Pax6 DNA-binding capacity and gene regulatory functions. Although this mutation did not result in gross morphological changes in amphioxus larvae, it demonstrated a reduced ability to activate Pax6-responsive reporter gene, suggesting a hypomorphic effect. Expression analysis in mutant larvae revealed changes in gene expression within the anterior CNS, supporting the conserved role of Pax6 gene in brain regionalization across chordates. Additionally, our findings lend support to the hypothesis of a zona limitans intrathalamica (ZLI)-like region in amphioxus, suggesting evolutionary continuity in brain patterning mechanisms. ZLI region, found in both hemichordates and vertebrates, functions as a key signaling center and serves as a restrictive boundary between major thalamic regions.

RESUMEN

Antimicrobial peptides (AMPs), characterized by their cationic nature and amphiphilic properties, play a pivotal role in inhibiting the biological activity of microbes. Currently, only a fraction of the antimicrobial potential within the ribosomal protein family has been explored, despite its extensive membership and resemblance to AMPs. Herein we demonstrated that amphioxus RPL17 (BjRPL17) exhibited not only upregulated expression upon bacterial stimulation but also possessed bactericidal capabilities against both Gram-negative and -positive bacteria through combined action mechanisms including interaction with cell surface molecules LPS, LTA, and PGN, disruption of cell membrane integrity, promotion of membrane depolarization, and induction of intracellular ROS production. Furthermore, a peptide derived from residues 127-141 of BjRPL17 (termed BjRPL17-1) showed antibacterial activity against Staphylococcus aureus and its methicillin-resistant strain via the same mechanism observed for the full-length protein. Additionally, the rpl17 gene was highly conserved in Metazoa, hinting it may play a universal role in the antibacterial defense system in different animals. Importantly, neither BjRPL17 nor peptide BjRPL17-1 exhibited toxicity towards mammalian cells thereby offering prospects for designing novel AMP agents based on these findings. Collectively, our results establish RPL17 as a novel member of AMPs with remarkable evolutionary conservation.

Asunto(s)

RESUMEN

Genomes and transcriptomes from diverse organisms are providing a wealth of data to explore the evolution and origin of neuropeptides and their receptors in metazoans. While most neuropeptide-receptor systems have been extensively studied in vertebrates, there is still a considerable lack of understanding regarding their functions in invertebrates, an extraordinarily diverse group that account for the majority of animal species on Earth. Cephalochordates, commonly known as amphioxus or lancelets, serve as the evolutionary proxy of the chordate ancestor. Their key evolutionary position, bridging the invertebrate to vertebrate transition, has been explored to uncover the origin, evolution, and function of vertebrate neuropeptide systems. Amphioxus genomes exhibit a high degree of sequence and structural conservation with vertebrates, and sequence and functional homologues of several vertebrate neuropeptide families are present in cephalochordates. This review aims to provide a comprehensively overview of the recent findings on neuropeptides and their receptors in cephalochordates, highlighting their significance as a model for understanding the complex evolution of neuropeptide signaling in vertebrates.

Asunto(s)

RESUMEN

Our understanding of the evolutionary origin of Chordata, one of the most disparate and ecologically significant animal phyla, is hindered by a lack of unambiguous stem-group relatives. Problematic Cambrian fossils that have been considered as candidate chordates include vetulicolians,1Yunnanozoon,2 and the iconic Pikaia.3 However, their phylogenetic placement has remained poorly constrained, impeding reconstructions of character evolution along the chordate stem lineage. Here we reinterpret the morphology of Pikaia, providing evidence for a gut canal and, crucially, a dorsal nerve cord-a robust chordate synapomorphy. The identification of these structures underpins a new anatomical model of Pikaia that shows that this fossil was previously interpreted upside down. We reveal a myomere configuration intermediate between amphioxus and vertebrates and establish morphological links between Yunnanozoon, Pikaia, and uncontroversial chordates. In this light, we perform a new phylogenetic analysis, using a revised, comprehensive deuterostome dataset, and establish a chordate stem lineage. We resolve vetulicolians as a paraphyletic group comprising the earliest diverging stem chordates, subtending a grade of more derived stem-group chordates comprising Yunnanozoon and Pikaia. Our phylogenetic results reveal the stepwise acquisition of characters diagnostic of the chordate crown group. In addition, they chart a phase in early chordate evolution defined by the gradual integration of the pharyngeal region with a segmented axial musculature, supporting classical evolutionary-developmental hypotheses of chordate origins4 and revealing a "lost chapter" in the history of the phylum.

Asunto(s)

RESUMEN

Growth hormone-releasing hormone (GHRH) has been widely shown to stimulate growth hormone (GH) production via binding to GHRH receptor GHRHR in various species of vertebrates, but information regarding the functional roles of GHRH and GHRHR in the protochordate amphioxus remains rather scarce. We showed here that two mature peptides, BjGHRH-1 and BjGHRH-2, encoded by BjGHRH precursor, and a single BjGHRHR protein were identified in the amphioxus Branchiostoma. japonicum. Like the distribution profiles of vertebrate GHRHs and GHRHRs, both the genes Bjghrh and Bjghrhr were widely expressed in the different tissues of amphioxus, including in the cerebral vesicle, Hatschek's pit, neural tube, gill, hepatic caecum, notochord, testis and ovary. Moreover, both BjGHRH-1 and BjGHRH-2 interacted with BjGHRHR, and triggered the cAMP/PKA signal pathway in a dose-dependent manner. Importantly, BjGHRH-1 and BjGHRH-2 were both able to activate the expression of GH-like gene in the cells of Hatschek's pit. These indicate that a functional vertebrate-like GHRH-GHRHR axis had already emerged in amphioxus, which is a seminal innovation making physiological divergence including reproduction, growth, metabolism, stress and osmoregulation possible during the early evolution of vertebrates.

Asunto(s)

RESUMEN

The mechanism of sex determination and differentiation in animals remains a central focus of reproductive and developmental biology research, and the regulation of sex differentiation in amphioxus remains poorly understood. Cytochrome P450 Family 19 Subfamily A member 1 (CYP19A1) is a crucial sex differentiation gene that catalyzes the conversion of androgens into estrogens. In this study, we identified two aromatase-like genes in amphioxus: cyp19-like1 and cyp19-like2. The cyp19-like1 is more primitive and may represent the ancestral form of cyp19 in zebrafish and other vertebrates, while the cyp19-like2 is likely the result of gene duplication within amphioxus. To gain further insights into the expression level of these two aromatase-like, we examined their expression in different tissues and during different stages of gonad development. While the expression level of the two genes differs in tissues, both are highly expressed in the gonad primordium and are primarily localized to microsomal membrane systems. However, as development proceeds, their expression level decreases significantly. This study enhances our understanding of sex differentiation mechanisms in amphioxus and provides valuable insights into the formation and evolution of sex determination mechanisms in vertebrates.

RESUMEN

C-type lectin proteins (CTLs), a group of pattern recognition receptors (PRRs), play pivotal roles in immune responses. However, the signal transduction and regulation of CTLs in cephalochordates have yet to be explored. In this study, we examined the composition of CTLs in Branchiostoma japonicum, identifying a total of 272 CTLs. These CTLs underwent further analysis concerning domain arrangement, tandem and segmental duplication events. A multidomain C-type lectin gene, designated as BjCTL5, encompassing CLECT, KR, CUB, MAM, and SR domains, was the focal point of our investigation. BjCTL5 exhibits ubiquitous expression across all detected tissues and is responsive to stimulation by LPS, mannose, and poly (I:C). The recombinant protein of BjCTL5 can bind to Escherichia coli and Staphylococcus aureus, inducing their agglutination and inhibiting the proliferation of S. aureus. Yeast two-hybrid, CoIP, and confocal immunofluorescence experiments revealed the interaction between BjCTL5 and apoptosis-stimulating proteins of p53, BjASPP. Intriguingly, BjCTL5 was observed to induce the luciferase activity of the NF-κB promoter in HEK293T cells. These results suggested a potential interaction between BjCTL5 and BjASPP, implicating that they involve in the activation of the NF-κB signaling pathway, which provides an evolutionary viewpoint on NF-κB signaling pathway in primitive chordate.

Asunto(s)

RESUMEN

Cystatins comprise a vast superfamily of evolutionary conserved proteins, predominantly recognized for their roles as endogenous inhibitors by regulating the activity of cysteine proteases. Emerging lines of research evidence also provides insight into their alternative roles in a spectrum of biological and pathological processes, including neurodegenerative disorders, tumor progression, inflammatory diseases, and immune response. Nowadays, various type-1 cystatins (stefins) have been demonstrated among a variety of discovered vertebrate groups, while little is known about the related homologue in cephalochordate amphioxus, which are repositioned at the base of the chordate phylum. In the present study, a single type-1 cystatin homologue in Branchiostoma japonicum was first successfully cloned and designated as Bjcystatin-1. The deduced Bjcystatin-1 protein is structurally characterized by the presence of typical wedge-shaped cystatin features, including the 'QxVxG' and 'Px' motif, as well as the conserved N-terminal glycine residue. Phylogenomic analyses utilizing different cystatin counterparts affirmed the close evolutionary relationship of Bjcystatin-1 and type-1 cystatin homologue. Bjcystatin-1 was predominantly expressed in the gills and hind-gut in a tissue-specific pattern, and its expression was remarkably up-regulated in response to challenge with bacteria or their signature molecules LPS and LTA, suggesting the involvement in immune response. Additionally, the recombinant Bjcystatin-1 (rBjcystatin-1) protein showed significant inhibitory activity towards papain and binding ability to LPS and LTA, indicating its hypothesized role as a pattern recognition receptor in immune response. Subcellular localization results also showed that Bjcystatin-1 was located in the cytoplasm and nucleus, and its overexpression could attenuate the activation of LPS-induced nuclear transcription factors NF-κB. Taken together, our study suggests that amphioxus Bjcystatin-1 acts as a dual role in protease inhibitor and an immunocompetent factor, providing new insights into the immune defense effect of type-1 cystatin in amphioxus.

Asunto(s)

RESUMEN

Cephalochordates occupy a key phylogenetic position for deciphering the origin and evolution of chordates, since they diverged earlier than urochordates and vertebrates. The notochord is the most prominent feature of chordates. The amphioxus notochord features coin-shaped cells bearing myofibrils. Notochord-derived hedgehog signaling contributes to patterning of the dorsal nerve cord, as in vertebrates. However, properties of constituent notochord cells remain unknown at the single-cell level. We examined these properties using Iso-seq analysis, single-cell RNA-seq analysis, and in situ hybridization (ISH). Gene expression profiles broadly categorize notochordal cells into myofibrillar cells and non-myofibrillar cells. Myofibrillar cells occupy most of the central portion of the notochord, and some cells extend the notochordal horn to both sides of the ventral nerve cord. Some notochord myofibrillar genes are not expressed in myotomes, suggesting an occurrence of myofibrillar genes that are preferentially expressed in notochord. On the other hand, non-myofibrillar cells contain dorsal, lateral, and ventral Müller cells, and all three express both hedgehog and Brachyury. This was confirmed by ISH, although expression of hedgehog in ventral Müller cells was minimal. In addition, dorsal Müller cells express neural transmission-related genes, suggesting an interaction with nerve cord. Lateral Müller cells express hedgehog and other signaling-related genes, suggesting an interaction with myotomes positioned lateral to the notochord. Ventral Müller cells also expressed genes for FGF- and EGF-related signaling, which may be associated with development of endoderm, ventral to the notochord. Lateral Müller cells were intermediate between dorsal/ventral Müller cells. Since vertebrate notochord contributes to patterning and differentiation of ectoderm (nerve cord), mesoderm (somite), and endoderm, this investigation provides evidence that an ancestral or original form of vertebrate notochord is present in extant cephalochordates.

Asunto(s)

RESUMEN

A central goal of evolutionary developmental biology is to decipher the evolutionary pattern of gene regulatory networks (GRNs) that control embryonic development, and the mechanism underlying GRNs evolution. The Nodal signaling that governs the body axes of deuterostomes exhibits a conserved GRN orchestrated principally by Nodal, Gdf1/3, and Lefty. Here we show that this GRN has been rewired in cephalochordate amphioxus. We found that while the amphioxus Gdf1/3 ortholog exhibited nearly no embryonic expression, its duplicate Gdf1/3-like, linked to Lefty, was zygotically expressed in a similar pattern as Lefty. Consistent with this, while Gdf1/3-like mutants showed defects in axial development, Gdf1/3 mutants did not. Further transgenic analyses showed that the intergenic region between Gdf1/3-like and Lefty could drive reporter gene expression as that of the two genes. These results indicated that Gdf1/3-like has taken over the axial development role of Gdf1/3 in amphioxus, possibly through hijacking Lefty enhancers. We finally demonstrated that, to compensate for the loss of maternal Gdf1/3 expression, Nodal has become an indispensable maternal factor in amphioxus and its maternal mutants caused axial defects as Gdf1/3-like mutants. We therefore demonstrated a case that the evolution of GRNs could be triggered by enhancer hijacking events. This pivotal event has allowed the emergence of a new GRN in extant amphioxus, presumably through a stepwise process. In addition, the co-expression of Gdf1/3-like and Lefty achieved by a shared regulatory region may have provided robustness during body axis formation, which provides a selection-based hypothesis for the phenomena called developmental system drift.

Asunto(s)

RESUMEN

In 1830, Cuvier and Geoffroy Saint-Hilaire confronted each other in a famous debate on the unity of the animal kingdom, which permeated the zoology of the 19th century. From that time, a growing number of naturalists attempted to understand the large-scale relationships among animals. And among all the questions, that of the origin of vertebrates was one of the most controversial. Analytical methods based on comparative anatomy, embryology and paleontology were developed to identify convincing homologies that would reveal a logical sequence of events for the evolution of an invertebrate into the first vertebrate. Within this context, several theories have clashed on the question of the identity of the ancestor of vertebrates. Among the proposals, a group of rather discrete organisms, the ascidians, played a central role. Because he had discovered an ascidian with a particularly atypical larval development, the Molgula, Henri de Lacaze-Duthiers, a rigorous and meticulous naturalist, became involved in the ascidian hypothesis. While the visionary mind of Lacaze-Duthiers led him to establish a particularly innovative methodology and the first marine biology station in Europe, at Roscoff, the tailless tadpole of the Molgula prevented him from recognizing the ancestor of vertebrates. This old 19th century story echoes the ever-present questions driving the field of Eco-Evo-Devo.

Asunto(s)

RESUMEN

Cephalochordates and tunicates represent the only two groups of invertebrate chordates, and extant cephalochordates - commonly known as amphioxus or lancelets - are considered the best proxy for the chordate ancestor, from which they split around 520 million years ago. Amphioxus has been an important organism in the fields of zoology and embryology since the 18th century, and the morphological and genomic simplicity of cephalochordates (compared to vertebrates) makes amphioxus an attractive model for studying chordate biology at the cellular and molecular levels. Here we describe the life cycle of amphioxus, and discuss the natural histories and habitats of the different species of amphioxus. We also describe their use as laboratory animal models, and discuss the techniques that have been developed to study different aspects of amphioxus.

Asunto(s)

RESUMEN

The mechanisms underlying the toxicity of environmental stress are unclear for marine macrobenthos. Copper/Cu has posed the most serious threats to amphioxus, an ancient and model benthic cephalochordate. Herein, a dynamic change in the physiological parameters (GR, SOD, ATP, and MDA) was detected with ROS accumulation in Branchiostoma belcheri exposed to 0.3 mg·L-1 Cu. Transcriptomes and microRNAomes of B. belcheri were generated to investigate the molecular mechanisms by which this amphioxus copes with Cu exposure. Time-specific genes identified at different time points after exposure were involved in the stimulus and immune response, detoxification and ionic homeostasis, aging and the nervous system, sequentially, with prolongation of exposure time, forming a dynamic process of molecular response to Cu stress. In total, 57 differentially expressed miRNAs were identified under Cu stress. Transcriptomics-miRNAomics analyses indicate that these miRNAs targeted genes associated with many key biological processes such as xenobiotics degradation, oxidative stress, and energy metabolism. The constructed miRNA-mRNA-pathway network uncovered a broad post-transcriptional regulatory mechanism in B. belcheri to cope with Cu stress. Overall, this integrated analyses show that enhanced defense response, accelerated ROS elimination, and repressed ATP production constitute a comprehensive strategy to cope with Cu toxicity in the ancient macrobenthos.

Asunto(s)

RESUMEN

Amphioxus species are considered living fossils and are important in the evolutionary study of chordates and vertebrates. To explore viral homologous sequences, a high-quality annotated genome of the Beihai amphioxus (Branchiostoma belcheri beihai) was examined using virus sequence queries. In this study, 347 homologous fragments (HFs) of viruses were identified in the genome of B. belcheri beihai, of which most were observed on 21 genome assembly scaffolds. HFs were preferentially located within protein-coding genes, particularly in their CDS regions and promoters. A range of amphioxus genes with a high frequency of HFs is proposed, including histone-related genes that are homologous to the Histone or Histone H2B domains of viruses. Together, this comprehensive analysis of viral HFs provides insights into the neglected role of viral integration in the evolution of amphioxus.

Asunto(s)

RESUMEN

Small ubiquitin-like modifier (SUMO) regulates various biological processes, including the MyD88/TICAMs-IRAKs-TRAF6-NF-κB pathway, one of the core immune pathways. However, its functions are inconsistent between invertebrates and vertebrates and have rarely been investigated in lower chordates, including amphioxus and fishes. Here, we investigated the SUMOylation gene system in the amphioxus, a living basal chordate. We found that amphioxus has a SUMOylation system that has a complete set of genes and preserves several ancestral traits. We proceeded to study their molecular functions using the mammal cell lines. Both amphioxus SUMO1 and SUMO2 were shown to be able to attach to NF-κB Rel and to inhibit NF-κB activation by 50-75% in a dose-dependent fashion. The inhibition by SUMO2 could be further enhanced by the addition of the SUMO E2 ligase UBC9. In comparison, while human SUMO2 inhibited RelA, human SUMO1 slightly activated RelA. We also showed that, similar to human PIAS1-4, amphioxus PIAS could serve as a SUMO E3 ligase and promote its self-SUMOylation. This suggests that amphioxus PIAS is functionally compatible in human cells. Moreover, we showed that amphioxus PIAS is not only able to inhibit NF-κB activation induced by MyD88, TICAM-like, TRAF6 and IRAK4 but also able to suppress NF-κB Rel completely in the presence of SUMO1/2 in a dose-insensitive manner. This suggests that PIAS could effectively block Rel by promoting Rel SUMOylation. In comparison, in humans, only PIAS3, but not PIAS1/2/4, has been reported to promote NF-κB SUMOylation. Taken together, the findings from amphioxus, together with those from mammals and other species, not only offer insights into the functional volatility of the animal SUMO system, but also shed light on its evolutionary transitions from amphioxus to fish, and ultimately to humans.

Asunto(s)

RESUMEN

The basal chordate amphioxus is a model for tracing the origin and evolution of vertebrate immunity. To explore the evolution of immunoreceptor signaling pathways, we searched the associated receptors of the amphioxus Branchiostoma belcheri (Bb) homolog of immunoreceptor signaling adaptor protein Grb2. Mass-spectrum analysis of BbGrb2 immunoprecipitates from B. belcheri intestine lysates revealed a folate receptor (FR) domain- and leucine-rich repeat (LRR)-containing protein (FrLRR). Sequence and structural analysis showed that FrLRR is a membrane protein with a predicted curved solenoid structure. The N-terminal Fr domain contains very few folate-binding sites; the following LRR region is a Slit2-type LRR, and a GPI-anchored site was predicted at the C-terminus. RT-PCR analysis showed FrLRR is a transcription-mediated fusion gene of BbFR-like and BbSlit2-N-like genes. Genomic DNA structure analysis implied the B. belcheri FrLRR gene locus and the corresponding locus in Branchiostoma floridae might be generated by exon shuffling of a Slit2-N-like gene into an FR gene. RT-qPCR, immunostaining, and immunoblot results showed that FrLRR was primarily distributed in B. belcheri intestinal tissue. We further demonstrated that FrLRR localized to the cell membrane and lysosomes. Functionally, FrLRR mediated and promoted bacteria-binding and phagocytosis, and FrLRR antibody blocking or Grb2 knockdown inhibited FrLRR-mediated phagocytosis. Interestingly, we found that human Slit2-N (hSlit2-N) also mediated direct bacteria-binding and phagocytosis which was inhibited by Slit2-N antibody blocking or Grb2 knockdown. Together, these results indicate FrLRR and hSlit2-N may function as phagocytotic-receptors to promote phagocytosis through Grb2, implying the Slit2-N-type-LRR-containing proteins play a role in bacterial binding and elimination.

Asunto(s)

RESUMEN

The slow-evolving invertebrate amphioxus has an irreplaceable role in advancing our understanding of the vertebrate origin and innovations. Here we resolve the nearly complete chromosomal genomes of three amphioxus species, one of which best recapitulates the 17 chordate ancestor linkage groups. We reconstruct the fusions, retention, or rearrangements between descendants of whole-genome duplications, which gave rise to the extant microchromosomes likely existed in the vertebrate ancestor. Similar to vertebrates, the amphioxus genome gradually establishes its three-dimensional chromatin architecture at the onset of zygotic activation and forms two topologically associated domains at the Hox gene cluster. We find that all three amphioxus species have ZW sex chromosomes with little sequence differentiation, and their putative sex-determining regions are nonhomologous to each other. Our results illuminate the unappreciated interspecific diversity and developmental dynamics of amphioxus genomes and provide high-quality references for understanding the mechanisms of chordate functional genome evolution.

Asunto(s)

RESUMEN

BACKGROUND: Amphioxus are non-vertebrate chordates characterized by a slow morphological and molecular evolution. They share the basic chordate body-plan and genome organization with vertebrates but lack their 2R whole-genome duplications and their developmental complexity. For these reasons, amphioxus are frequently used as an outgroup to study vertebrate genome evolution and Evo-Devo. Aside from whole-genome duplications, genes continuously duplicate on a smaller scale. Small-scale duplicated genes can be found in both amphioxus and vertebrate genomes, while only the vertebrate genomes have duplicated genes product of their 2R whole-genome duplications. Here, we explore the history of small-scale gene duplications in the amphioxus lineage and compare it to small- and large-scale gene duplication history in vertebrates. RESULTS: We present a study of the European amphioxus (Branchiostoma lanceolatum) gene duplications thanks to a new, high-quality genome reference. We find that, despite its overall slow molecular evolution, the amphioxus lineage has had a history of small-scale duplications similar to the one observed in vertebrates. We find parallel gene duplication profiles between amphioxus and vertebrates and conserved functional constraints in gene duplication. Moreover, amphioxus gene duplicates show levels of expression and patterns of functional specialization similar to the ones observed in vertebrate duplicated genes. We also find strong conservation of gene synteny between two distant amphioxus species, B. lanceolatum and B. floridae, with two major chromosomal rearrangements. CONCLUSIONS: In contrast to their slower molecular and morphological evolution, amphioxus' small-scale gene duplication history resembles that of the vertebrate lineage both in quantitative and in functional terms.

Asunto(s)

RESUMEN

The core molecular mechanisms of dorsal organizer formation during gastrulation are highly conserved within the chordate lineage. One of the key characteristics is that Nodal signaling is required for the organizer-specific gene expression. This feature appears to be ancestral, as evidenced by the presence in the most basally divergent chordate amphioxus. To provide a better understanding of the evolution of organizer-specific gene regulation in chordates, we analyzed the cis-regulatory sequence of amphioxus Chordin in the context of the vertebrate embryo. First, we generated stable zebrafish transgenic lines, and by using light-sheet fluorescent microscopy, characterized in detail the expression pattern of GFP driven by the cis-regulatory sequences of amphioxus Chordin. Next, we performed a 5'deletion analysis and identified an enhancer sufficient to drive the expression of the reporter gene into a chordate dorsal organizer. Finally, we found that the identified enhancer element strongly depends on Nodal signaling, which is consistent with the well-established role of this pathway in the regulation of the expression of dorsal organizer-specific genes across chordates. The enhancer identified in our study may represent a suitable simple system to study the interplay of the evolutionarily conserved regulatory mechanisms operating during early chordate development.

Asunto(s)