Ugo Coppola

Craniofacial malformations arise from developmental defects in the head, face, and neck with phenotypes such as 22q11.2 deletion syndrome illustrating a developmental link between cardiovascular and craniofacial morphogenesis. NKX2-5 is a key cardiac transcription factor associated with congenital heart disease and mouse models of Nkx2-5 deficiency highlight roles in cardiac development. In zebrafish, nkx2.5 and nkx2.7 are paralogues in the NK4 family expressed in cardiomyocytes and pharyngeal arches. Despite shared cellular origins of cardiac and craniofacial tissues, the function of NK4 factors in head and neck patterning has not been elucidated. Molecular evolutionary analysis of NK4 genes shows that nkx2.5 and nkx2.7 are ohnologs resulting from whole genome duplication events. Nkx2.7 serves as a previously unappreciated regulator of branchiomeric muscle and cartilage formation for which nkx2.5 cannot fully compensate. Mechanistically, our results highlight that Nkx2.7 patterns the cranial neural crest and functions upstream of Endothelin1 to inhibit Notch signals. Together, our studies shed light on an evolutionarily conserved Nkx transcription factor with unique functions in vertebrate craniofacial development, advancing our understanding of congenital head and neck deformities.

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.

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.

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. 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. 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.

Nitric oxide synthase is ubiquitously present in metazoans and is involved in a wide range of biological processes. Three distinctNosgenes have been so far identified in vertebrates exhibiting a complex expression pattern and transcriptional regulation. Nevertheless, although independent events ofNosduplication 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-typeNosexpression, we focused on two non-vertebrate chordates: the cephalochordateBranchiostoma lanceolatumand the tunicateCiona robusta. Here, throughout transphyletic and transgenic approaches, we identified genomic regions in both species acting asNosfunctional enhancers during development.In vivoanalyses ofNosgenomic fragments revealed their ability to recapitulate the endogenous expression territories. Therefore, our results suggest the existence of evolutionary conserved mechanisms responsible for neuronal-typeNosregulation in non-vertebrate chordates. In conclusion, this study paves the way for future characterization of conserved transcriptional logic underlying the expression of neuronal-typeNosgenes in chordates.

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.

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.

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.

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 Glade. 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.