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.

In vertebrates, Uncx is transcribed in sclerotomal cells surrounding the notochord, suggesting a conserved role as determinant of axial skeleton morphogenesis (Neidhardt et al., 1997;Mansouri et al., 1997;Koudijs et al., 2008;Sánchez and Sánchez, 2013;Retnoaji et al., 2014). Uncx functions are perhaps best understood in amniotes. Loss-of-function studies in mice support a role in the condensation of mesenchymal cells of the lateral sclerotome and proper development of pedicles, transverse processes, and proximal rib derivatives. Moreover, disruption to the establishment of antero-posterior (AP)-somite polarity in Uncx mutant mice suggests that this gene is required for the maintenance of posterior somite characteristics (Leitges et al., 2000;Mansouri et al., 2000).
Uncx transcription in the presomitic mesoderm (PSM) depends on Delta-like 1 (Dll1) and is independent from signals of the axial structures, such as notochord-floor plate complex, whereas further maintenance requires Uncx itself (Barrantes et al., 1999;Mansouri et al., 2000;Schrägle et al., 2004;Sewell et al., 2009). A central role in the repression of Uncx expression in the anterior somite is played by a complex regulatory network that involves the basic helix-loop-helix transcription factor Mesp2, its downstream co-repressor Ripply, the homeodomain transcription factor MEOX1, and a cross-negative regulation with the T-box protein Tbx18 (Takahashi et al., 2000(Takahashi et al., , 2003(Takahashi et al., , 2013Nakajima et al., 2006;Farin et al., 2008;Skuntz et al., 2009;Yabe et al., 2016). Recently, cell type-specific epigenetic regulation of Uncx gene expression has been associated with axon guidance in C. elegans (Zheng et al., 2013) and with human leukemia (Daniele et al., 2017). It has been proposed that Uncx is implicated in cell cycle progression of neuronal progenitor cells, survival of olfactory epithelium and differentiation of dopaminergic neurons (Sammeta et al., 2010;Rabe et al., 2012).
Although many advances have been made in dissecting the biological significance for development and the mechanisms of action of vertebrate Uncx, other aspects, including molecular evolution and roles in axonal growth, remain poorly defined. To elucidate the cascade of events accomplished by the Uncx proteins the zebrafish (Danio rerio) could be an ideal model due to its amenability to embryological and genetic approaches. However, to date Uncx homologs in zebrafish have not been characterized in detail. In this study, we performed genome and gene expression analyses of the zebrafish genes uncx4.1 and uncx, with a focus on somite formation and innervation. Taken together, our results provide insights into the potential role of zebrafish uncx genes in the formation of spatially distinct muscle progenitor domains and in axon pathfinding.

Molecular evolution
The protein sequences used for the evolutionary analysis were retrieved from the NCBI and Ensembl databases. The Homo sapiens UNCX protein was the initial query sequence employed for tBlastn searches (Gertz et al., 2006) in invertebrate and vertebrate genomes, and reciprocal Blasts were carried out on each genome. ClustalW was used to align the proteins selected for phylogenetic analysis with default parameters (Thompson et al., 1994). The phylogenetic tree was built with the Maximum-Likelihood estimation (MLE) using MEGA6 with 1000 replicates; the LG substitution model, with 0.2 as proportion of invariable sites (I) and 4 as gamma distribution parameter (γ), was selected (Tamura et al., 2013). The graphical representation was created with Dendroscope (Huson and Scornavacca, 2012). The synteny analysis between human and zebrafish chromosomes was performed with "Sinteny Database" and a sliding window size of 50 genes (Catchen et al., 2009). The syntenic survey between human and the tunicate Ciona robusta was performed mapping manually the genes on the scaffolds/chromosomes in Ensembl and Genomicus databases. Introns were mapped by using available public resources and designed, with a color code representation, on the protein alignment obtained using ClustalW. The analysis of genomic conservation was performed on ten sequences, employing mVISTA computational tool (Ratnere and Dubchak, 2009). To identify conserved non-coding sequences by VISTA, we employed LAGAN (global pair-wise and multiple alignments of finished sequences) with the following parameters: minimum Conservation Width for non-coding sequences (40 bp), minimum conservation identity (50%), and minimum Y value (20%). To improve the comparison of distant homologs, the translated anchoring in LAGAN/ Shuffle-LAGAN was used.

Actin filament staining
Whole-mount phalloidin staining was performed as described (Whitfield et al., 1996). Embryos were fixed with 4% paraformaldehyde (PFA), followed by permeabilization in 2% Triton X-100/PBS for 1.5 h and incubation in 2.5 μg/ml fluorescein-labeled phalloidin (Sigma) in PBS for 2 h in the dark at 4°C. Embryos were then rinsed overnight and mounted in 70% glycerol/30% PBS prior to proceed to image acquisition.

Cloning and probe synthesis
A neurula stage zebrafish cDNA library (kind gift of D. Grunwald) prepared in the λ ZAP II vector (Stratagene) was screened for homeobox-containing genes by PCR with a primer annealing to the cloning site of the plasmid vector and a degenerate primer annealing to a conserved homeobox region (TTGACCCKCCKGTTYTGRAACCA). We cloned a 220 bp cDNA fragment of uncx4.1 that was used as probe to screen 1.2/10 6 recombinant plaques of the same library at moderate stringency. From a fourth screen, a Bluescript phagemid was rescued and its 2 kb insert sequenced, which encoded a full-length Uncx4.1 protein as judged by a BLAST search of GenBank and EMBL databases. To make riboprobes for WISH analysis, a 950 bp uncx4.1-containing pBluescript SK+ plasmid was linearized with EcoRI and transcribed with T7 for the antisense probe, or linearized with ApaI and transcribed using T3 polymerase for the sense riboprobe. A 562 bp cDNA fragment of the uncx gene coding sequence was amplified (Fwd: 5′-AGCCACCA TCATGTGTACGA-3′ and Rev: 5′-CGGGAAGGAGTTTGTTTTGA-3′), cloned into pCR™ II-TOPO® vector following TOPO TA Cloning instruction manual, and sequenced. The TOPO TA plasmid was linearized with HindIII and transcribed using T7 polymerase for the antisense probe or linearized with ApaI and transcribed using SP6 polymerase for the sense probe (Suppl. Fig. 1).

Microinjections
mRNA: To generate synthetic mRNA for injection, the entire uncx4.1 reading frame (ORF) was cloned into the vector pβUT2 which was made by cloning 5′ and 3′ UTRs of Xenopus β-globin (from pSP64T; Krieg and Melton, 1984) at either side of pBlueScript (Stratagene) with a synthetic polylinker replacing the BglII site of pSP64T. To clone the uncx4.1 gene ORF in-frame with the Kozak consensus sequence, which increases the efficiency of translation initiation by ribosomes (Kozak, 1986), a PCR on the 2 kb uncx4.1 insert (see Section 2.4) was performed using the following primers: GACGAAGGTACCCCACCATGATGGATAG CCGGATC and CCTATTGGTACCTCAGTGCATGTCTACATC. The 1340 bp PCR fragment was gel purified, cut with KpnI (introduced into the sequence through the primers), and ligated into the KpnI-digested pβUT2 plasmid. The DNA sequence of the insert was confirmed by sequencing. The plasmid was linearized with EcoRI and the gene was transcribed in vitro with the help of the T3 mMessage mMachine Kit (Ambion) yielding capped RNA for injection. The zebrafish full-length shha-containing pSP64T plasmid for mRNA injection was a kind gift of P. Ingham (Krauss et al., 1993). Synthetic capped shha and uncx4.1 mRNAs were injected repeatedly (n > 3) at concentrations of 400, 200, and 200 pg per embryo, respectively. Injections were carried out on 1-to 2-cell stage embryos.
The study of the Uncx genomic locus revealed a high degree of synteny between tetrapods as human (Chr7) and teleosts as zebrafish (Chr1, Chr3), showing the preservation of 11 genes close to Uncx: the conservation of this cluster supports a TSGD-origin for zebrafish Uncx genes. The absence of some of these genes (e.g., gper) in one of the two syntenic clusters suggests the secondary loss of TSGD-derived duplicates (Fig. 2).
Our survey also expanded our understanding of conservation of the Uncx genomic locus (Woolfe and Elgar, 2007), demonstrating high synteny in gnathostomes. In particular, we uncovered the presence of two conserved microsyntenic clusters. First, a gene triplet composed of Uncx, Micall2 and Elfn1 genes is present in gnathostomes. Mical The complete protein sequences were employed for tree inference. Uncx proteins deriving from teleost-specific genome duplication (TSGD) are shown in red. Protostome and Ambulacrarian Uncx proteins are grouped in the blue and green triangle, respectively. All sequences used in this analysis are reported in the Suppl. File 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) (molecule interacting with CasL) and Micall are cytosolic multidomain proteins that have been associated to axon guidance, cell movement, cell-cell junction formation, vesicle trafficking, and cancer cell metastasis (Xue et al., 2010). Elfn1 (Extracellular Leucine Rich Repeat And Fibronectin Type III Domain Containing 1) is a protein specifically present in excitatory synapses, where it acts as a regulator of presynaptic release probably to direct interneuron recruitment (Cao et al., 2015). Despite the absence of synteny between Olfactores (Tunicata and Vertebrata) and other Metazoans, we traced back a gene duplet formed by Uncx and Elfn1 genes in C. robusta (Chr11) and H. sapiens (Chr7) genomes (Suppl. Fig. 2). Importantly, this gene pair has been retained in all gnathostomes (data not shown). Concerning invertebrates, we also found two Uncx genes on the same chromosomal region in D. melanogaster, C. teleta, and S. kowalevskii (Suppl. Fig. 3). This Uncx duplet is flanked by Alx, which encodes a transcription factor with chondrogenic and other functions in vertebrates (Gordon et al., 1996), whereas Alx has been lost in Drosophila (Ryan et al., 2006). Furthermore, the amphioxus B. floridae ortholog clustered with the homeobox Rx gene (Irimia et al., 2012), essential for eye development (Sinn and Wittbrodt, 2013) (Suppl. Fig. 3).
Next, we sought to study the genomic region between the Uncx and Micall2 genes, a duplet present only in gnathostomes and that has undergone duplication in teleosts (uncx4.1-uncx, micall2a-micall2b) (Fig. 2). A VISTA analysis revealed some conserved peaks within this gene duplet (Suppl. Fig. 4). Then, we analyzed the genomic locus of Uncx in ten metazoans selecting ca. 3000 base pairs downstream and upstream of the Uncx orthologous genes (Suppl. Fig. 5). Our plot revealed a conserved upstream region, whose traces are visible also in C. gigas, in L. anatina and C. robusta. With respect to vertebrates, teleosts did not show plain conservation in the upstream region (orange box) and in the second intron (green box). Notably, the upstream peak pattern differs between teleost paralogs (blue box). Altogether, this indepth study of Uncx evolution defines this gene as an ultra-conserved homeobox gene with a complex evolutionary scenario in vertebrates.

Uncx expression during embryonic development
To generate probes for mRNA in situ hybridization, we first cloned the full-length transcripts of uncx4.1 (NM_001020780) and uncx (XM_005164204.4). In order to have a spatio-temporal overview of uncx gene expression during zebrafish embryogenesis, we performed a whole-mount in situ hybridization (WISH) at various developmental stages until 48 hours post fertilization (hpf).
Recently, tomographic data based on low-input RNA sequencing and mathematical image reconstruction have revealed early expression of uncx4.1 in the shield at 5 hpf (Junker et al., 2014). In this study, the earliest evidence of uncx4.1 expression was detected in all but the most anterior cells within each somite (11 hpf) (Fig. 3A). During early somitogenesis, uncx4.1 expression is reiterated in newly forming metameric blocks (14.5 hpf) (Fig. 3B-D). Subsequently, expression disappears from the myoseptum to dorsal and ventral margins (19 hpf) (Fig. 3E) to become restricted in few boundary cells positioned in the ventral lateral posterior (VLP) tip of the somite (24-34 hpf) ( Fig. 3F-H, J). VLP cells can be easily recognized at the cellular level with differential interference contrast optics, due to the round cell shape compared to the elongated fibroblasts. Phalloidin staining of 34 hpf embryos supports the view that uncx4.1 transcripts mark cells of the future myomere that are morphologically distinct from the main adaxial somite portion (Fig. 3I). We also found uncx4.1-expressing cells on both sides of the notochord, perhaps corresponding to the fish sclerotome, the myogenic contribution to backbone formation (Morin-Kensicki and Eisen, 1997) (Fig. 3K, L).

Characterization of uncx4.1 expression during somite differentiation
To further explore uncx4.1 expression during somitogenesis, we carried out double WISH with her1 and mespaa, which revealed that the earliest sign of uncx4.1 expression occurs in the anterior margin of the PSM and in the anlage of newly forming somite (Fig. 6A, B) (Holley et al., 2000(Holley et al., , 2002Sawada et al., 2000). In paraxial mesoderm, the adaxial cells are the first muscle precursors to express the transcription factor encoding gene myod1. These cells migrate laterally to give rise to slow muscle fibers (Devoto et al., 1996). Double hybridization shows that uncx4.1 mRNA is absent from adaxial cells and is co-expressed with myod1 in non-adaxial muscle precursor cells at early somitogenesis stages (12 hpf, 6 s) (Fig. 6C-E). Co-labeling of uncx4.1 mRNA and a panmyosin antigen (MF20, myosin heavy chain) illustrated how the expansion of myosin signal in maturing muscle cells occurs at the expenses of uncx4.1 expression in muscle progenitor cells (Fig. 6F-H).
As a preliminary assessment of potential roles for Uncx genes in somitogenesis in zebrafish, we used morpholino (MO) mediated knockdown of the zebrafish uncx genes. Unfortunately, injected uncx4.1 and uncx MOs led to various abnormalities including severe defects in body morphology with defective somitogenesis. Consequently we analyzed muscle differentiation only in those MO-microinjected larvae that were not disturbed in their overall morphology (about 50% uncx4.1 morphants, 34% uncx morphants). Analysis of fast and slow muscle fibers in such 34 hpf embryos injected with transcriptional start site MOs and subsequently stained for F59 (fast and slow MyHCs) and S58 (slow MyHC2) immunostaining shows normally differentiated myoblast cell types in chevron-shaped somites (Suppl. Fig. 6). These results do not allow us to make strong conclusions on potential roles for the uncx genes in the somites.

Uncx expression and signaling pathways during somitogenesis
Previous evidence suggests that signaling gradients from the neural tube and notochord-floor plate control the dynamic expression of the Uncx gene during somitogenesis (Schrägle et al., 2004). We thus investigated the potential involvement of the Hedgehog, FGF, Notch/ Delta and Nodal pathways in directing the spatial expression of zebrafish Uncx genes.

Hedgehog
Sonic Hedgehog (Shh) is a signaling molecule secreted by the notochord and floor plate which transduces via two transmembrane proteins, Patched 1 (Ptc1) and Smoothened (Smu), and regulates the activity of cubitus interruptus-related (Gli) transcription factors (Borycki et al., 2000). In turn, Gli proteins may act as activators or repressors of Hh signaling (Karlstrom et al., 2003;Tyurina et al., 2005). In zebrafish, the sonic hedgehog-a (shha) gene is expressed in the notochord and floorplate of the neural tube (Krauss et al., 1993).
Our data indicate that uncx4.1 expression ceases in myogenic progenitor cells localized immediately next to shha-expressing tissues (Fig. 7A, B). Furthermore, microinjection of full-length shha mRNA abolishes uncx4.1 expression in zebrafish embryos (Fig. 7C, D), suggesting that Shh may act negatively on uncx4.1 expression. However, the analysis of several zebrafish mutant embryos in the Hh signaling pathway provides a contrasting view of the regulatory role played by Hh signals on uncx4.1 and uncx expression during somitogenesis. The mutant lines studied include sonic-you (syu tbx392 ; sonic hedgehog a, shha) (Brand et al., 1996), slow-muscle-omitted (smu b577 ; smoothened) (Varga et al., 2001), you-too (yot ty119 ; gli2) (Karlstrom et al., 1999), and floating head (flh n1 ; noto) (Melby et al., 1996), the latter mutant lacking shhaexpressing tissues, i.e. notochord and most of the floor plate. In syu embryos, uncx4.1 is still expressed in the myoseptum and dorsal somite cells at 19 and 30-34 hpf, respectively (compare Figs. 3E and 7E, and 3H and 7F). The effect of Shh loss in syu embryos seems to be more pronounced in early uncx expression, as documented by diffuse mRNA labeling in the ventral portion of the somites at 19 hpf (compare Fig. 5B and F with Fig. 7G). In smu embryos, the VLP domain of uncx4.1  (Fig. 7I, J). It has already been demonstrated that the level of ptc transcripts, a target of Hh signaling, drops by more than half when using 50 μM cyclopamine, a concentration sufficient to impair slow muscle cell differentiation (Wolff et al., 2003). In our hands, inhibition of Hh signaling with cyclopamine does not alter the spatial and temporal transcriptional dynamics of uncx4.1 and uncx when compared with control EtOH-treated embryos ( Fig. 7K-N). Finally, uncx4.1 expression in flh mutant embryos is expanded dorsally and medially along the somite posterior boundary with reference to sibling controls (Fig. 7O, P). Since the flh mutant lacks the entire notochord and most of the floor plate, this expansion may be the manifestation of a synergic action of different hh genes (i.e., shha, shhb, ihhb) (Halpern et al., 1995).

FGF
In the zebrafish, Fgf signaling promotes posterior mesoderm development and border positioning (Sawada et al., 2001). Acerebellar (ace, fgf8a) mutant embryos exhibit only mild somite defects (Reifers et al., 1998;Draper et al., 2003). Groves et al. (2005) have demonstrated that Fgf8a mediates the promotion of a lateral fast muscle fibre population in zebrafish somite. Fgf8a drives myod1 expression in the lateral posterior stripe of immature caudal somites and is required for the lateral terminal differentiation of fast fibers in maturing rostral somites (Groves et al., 2005).
fgf8a mutant embryos (acerebellar, ace ti282a ) (Reifers et al., 1998) were thus used to explore the possible contribution of FGF signaling in the regulation of uncx genes during somite development. Both uncx4.1 and uncx fail to confine ventrally in ace homozygote embryos, suggesting that Fgf8a negatively regulates the expression of uncx4.1 (compare Figs. 3H and 8A for uncx4.1; Figs. 5B and 8B for uncx). Accordingly, the expression of uncx in embryos exposed to the Fgf inhibitor SU5402 is disrupted and partially dorsally expanded compared with control embryos treated with DMSO vehicle (compare Fig. 8C with Fig. 8D).

Nodal
A conserved role for Nodal factors, belonging to the TGFβ family, has been proposed in the formation of the mesoderm (Harland and Gerhart, 1997;Hagos and Dougan, 2007). The dynamic expression of uncx4.1 and uncx was largely unchanged during somite formation in the cyclops mutant (cyc b16 ; nodal-related protein, ndr2) (Rebagliati et al., 1998) Fig. 3F with Fig. 8E, and Fig. 5H with Fig. 8F). Similarly, uncx expression was normal in embryos treated with SB431542, an inhibitor of Nodal signaling (compare Fig. 8C with Fig. 8G).

Notch/Delta
In chi9ck, it has been proposed that Notch/Delta signaling induces Uncx transcription in the cranial PSM (Schrägle et al., 2004). We aimed to verify if the regulatory interaction between Notch/Delta driven oscillator activity and Uncx gene expression suggested in birds is conserved in teleosts. Previous data indicate that pharmacological blockade of the Notch/Delta pathway in zebrafish, by using the gammasecretase inhibitor DAPT, induces somite defects only after long developmental delays, suggesting that Notch/Delta signaling is essential for synchronizing oscillations of neighboring cells in the posterior PSM but not for somite border formation (Mara et al., 2007;Özbudak and Lewis, 2008;Sewell et al., 2009). We found that the expression of uncx4.1 and uncx in zebrafish embryos treated with DAPT is similar to that observed in control DMSO-treated embryos (compare Fig. 8H with Fig. 8I, and compare Fig. 8C with Fig. 8J, respectively). 3.6. Uncx4.1 and axogenesis C. elegans Unc-4 is well known for its role in axonal connections, acting as a determinant of synaptic choice for motor neurons (Schneider et al., 2012). In this study, we observed ancient syntenic association between Uncx and two genes involved in axogenesis (a Mical gene, micall2) and synaptic choice (Elfn1). Looking for correlations between Uncx gene expression and axon guidance, we first performed double labeling with the primary motor axon marker znp1, finding that the outgrowth of the caudal primary (CaP) motor axons coincides with the progressive down-regulation of uncx4.1 expression during somite development (Suppl. Fig. 7A, B, G). Then, we observed that the netrin-1b (ntn1b) gene, a member of a secreted protein family mediating axon guidance, is expressed in VLP cells at 34 hpf (Suppl. Fig. 7C, D-F, and Fig. 3I with Suppl. Fig. 7F). Finally, uncx4.1 over-expressing embryos display marked up-regulation of ntn1b with stunted and prematurely branching CaP axons, a phenotype possibly caused by surrounding the motor neuron growth cone with cells ectopically expressing the chemoattractant ntn1b (Suppl. Fig. 7H-K).

Origin and evolution of the Uncx genes
Since Metazoan Uncx proteins are poorly characterized from an evolutionary perspective (Woolfe and Elgar, 2007;Sánchez and Sánchez, 2013), we provided a comprehensive phylogenetic reconstruction showing the orthology of all analyzed genes, which we refer to as Uncx (Fig. 1). The partial protein-coding sequence found in the cnidarian N. vectensis genome (Suppl. File 2) as well as the absence of Uncx in sponges (Porifera), suggest that this homeobox gene was already present in the ancestor of bilaterians, though with instances of gene duplication and/or gene loss (Fig. 2). A common origin for Uncx genes is confirmed by the conservation of intron/exon structure in Bilateria (Suppl. File 3). We highlighted duplications in unrelated invertebrate taxa (i.e., D. melanogaster, C. teleta, S. kowalevskii, C. robusta) and the absence of Uncx in early branching metazoans (e.g., Placozoa and Ctenophora), in the agnathan lamprey and in many reptiles, which may underly functional gene diversification, with loss or (re)gain of (ancestral) gene functions (Albalat and Cañestro, 2016). As proposed for T. rubripes (Woolfe and Elgar, 2007), we report that teleosts have two Uncx duplicates, currently known as Uncx4.1 and Uncx. The retention of both co-orthologs reflects the over-representation of duplicated transcription factors in fish genomes (Roest Crollius and Weissenbach, 2005), depending on key roles in development and cellular differentiation. Interestingly, two rounds of whole-genome duplications (WGDs) at the stem of vertebrates (Ohno, 1993;Abi-Rached et al., 2002;Dehal and Boore, 2005) imply the presence of other three Uncx members in gnathostome ancestor, which have been secondarily lost during evolution.
We sought to provide insights into Uncx molecular evolution by analysing its genomic locus from cnidarians to human. It has been reported the presence of almost 800 conserved ancestral microsyntenic pair (CAMP) combinations for several homeobox genes as Uncx from cnidarians as Nematostella to cephalochordates as Branchiostoma (Irimia et al., 2012). We found that the Uncx gene forms distinct microsyntenic clusters. An invertebrate CAMP with the transcription factor encoding Alx/Cart-1 gene is seen in annelids and hemichordates (Suppl. Fig. 3), while in surveyed Olfactores, Uncx orthologs are coupled with Elfn1 (Suppl. Fig. 2). A cluster formed by Uncx, Elfn1, and Micall2 genes exists in gnathostomes, which is also duplicated in teleosts (Fig. 2). The conserved chromosomal vicinity of Uncx and Micall2 genes evokes a "bystander interference effect" exerted by one of the two genes, which has been proposed for genes implicated in key developmental mechanisms (Cajiao et al., 2004).
VISTA comparison of Uncx loci belonging to mollusks, brachiopods, ascidians, and vertebrates indicates conservation of sequence, consistent with past studies on Uncx highlighting the presence of CNEs (conserved non-coding elements) in Takifugu rubripes and Homo sapiens (Woolfe and Elgar, 2007) (Suppl. Fig. 5). Teleost Uncx paralogs lack some of the conserved elements common among coelacanth, spotted gar and human, whose lineages diverged from teleosts before the TSGD (Suppl. Fig. 5). In addition, they exhibit differences in peak patterns as if had undergone an asymmetrical rate of evolution.
The expression patterns of the two Uncx paralogous genes show unique (uncx4.1: pharyngeal arches and kidney; uncx: spinal cord) and partially overlapping domains (CNS and somites). These findings are possibly associated with genome duplication producing divergent  Fig. 3H and I, respectively). V. Nittoli, et al. Gene: X 2 (2019) 100011 regulatory modality, with events of subfunctionalization and/or neofunctionalization. However, the potential for cross-hybridisation needs to be considered when working with paralogous genes. In our work, divergent hybridization patterns with high signal and low background riboprobes were obtained, indicative of high levels of specificity and minimal cross-hybridization between duplicated genes. The analysis of vertebrate genome environment demonstrated that Uncx4.1 and Uncx genes descend from the same paralogon (Fig. 2); therefore, they derive from the teleost-specific genome duplication (TSGD), which occurred 300-350 million of years ago (Taylor et al., 2001;Taylor et al., 2003;Jaillon et al., 2004;Kuraku and Meyer, 2009). In light of the above, we propose to change the name of teleost Uncx paralogs genes into uncxa (Uncx4.1; NM_001020780.2) and uncxb (uncx; XM_005164204.4).

Regulation of the zebrafish uncx genes
In this study, uncx4.1 gene co-expression in the anterior presomitic mesoderm (PSM) with Notch-pathway gene her1, the output of the molecular clock, and with the Mesp1-related factor encoding mespaa (Fig. 6A, B), suggests that zebrafish uncx genes are controlled by players in somite anterior-posterior specification. This observation also indicates that, similarly to what is observed in mouse, zebrafish Uncx genes could be required for maintaining antero-posterior polarity within the somite (Farin et al., 2008;Lee et al., 2009). However, it is worth to note that the murine Uncx gene is expressed in the posterior half of the newly formed somites but, unlike the fish and chick orthologs, it is not active in the PSM (Barrantes et al., 1999;Schrägle et al., 2004). Furthermore, we show that, as in the zebrafish mesp quadruple  (Yabe et al., 2016). Considering that mouse Tbx6 is involved in somite boundary positioning together with Mesp, and that Mesp provides positional information within the somite, a similar mechanism to induce zebrafish uncx gene expression in the caudal somite half may occur during the establishment of somite polarity and boundary formation. In zebrafish, the somite develops into a large myotome, with a smaller group of ventral cells specified as sclerotome . Genes encoding myogenic regulatory factors such as myod1 and myf5 are expressed early in the most medial presomitic mesoderm adjacent to the notochord (Devoto et al., 1996;Weinberg et al., 1996). Both myod and myf5 control the commitment to the myogenic lineage and are required for the initiation of the myogenin gene expression (Pownall et al., 2002). During early somitogenesis in zebrafish, uncx4.1 activation coincides with that of myod1 in muscle progenitor cells (Fig. 6C, D), indicating that zebrafish Uncx paralogues may function in somites at the onset of muscle differentiation. The absence of uncx4.1 and uncx gene expression in adaxial cell precursors adjacent to the notochord ( Fig. 6C-E) suggests that uncx genes are not required for the specification and differentiation of slow muscle cells.
During somite formation, the distribution of uncx4.1 and uncx transcripts becomes progressively confined to a small population of undifferentiated myoblasts at the ventral lateral posterior (VLP) margin (Figs. 3 and 6). VLP cells expressing Uncx genes are likely connected to an extended ventral monolayer termed growth zone, which is known to contribute to hyperplastic growth of each myotome in marine teleosts (Barresi et al., , 2001. In this view, uncx4.1 could inhibit muscle formation via induction of myoblast proliferation at the expenses of muscle differentiation and/or as an antagonist of late differentiation (Fig. 6F-H).
We attempted to place uncx genes in the context of signal transduction mechanisms (i.e., Hh, FGF, Notch/Delta, Nodal) already known to play key roles in somite patterning and differentiation in zebrafish. Hh signal transduction is an intricate molecular pathway that acts in a dosage-dependent manner to specify cell fate in the zebrafish myotome (Wolff et al., 2003). The expression of uncx4.1 is lost in the Hh pathway component ptc1; ptc2 mutants (Koudijs et al., 2008). However, our data do not clarify whether or not Hedgehog signaling is required to drive expression of uncx4.1 and uncx; and, if so, to which extent. Also, the analysis of the regulatory interactions between Uncx genes and the Notch/Delta and Nodal pathways do not provide conclusive results with only changes to uncx expression. Accordingly, previous evidence in Notch1 mutant mouse shows that Uncx expression is slightly wider than in sibling embryos but essentially unaltered (Barrantes et al., 1999). Finally, somite expression of zebrafish Uncx genes in Fgf8a mutant embryos and in embryos treated with the Fgf inhibitor SU5402 is disrupted and dorsally extended, consistent with a negative role played by Fgf signaling in the expression of Uncx genes in zebrafish somitogenesis. The relationship between Uncx expression and fast muscle fibers warrants more careful examination in zebrafish fgf8a mutants. When all our evidence is considered, it suggests a hypothesis whereby Uncx gene expression is specifically regulated by Fgf signaling, while Hh, Notch/ Delta and Nodal signals may have more subtle roles in controlling the dynamic pattern of Uncx expression during somitogenesis.

A dual role in somitogenesis and axon guidance?
In silico analysis of available genome databases revealed the physical co-localization of Uncx with genes implicated in synaptic functioning and plasticity, i.e. Micall2 (gnathostome-specific gene duplet). Also, a correlation was observed between the expression patterns of uncx4.1 and ntn1b, a member of a secreted protein family mediating axon guidance, and the trajectory of caudal primary (CaP) motor neuron axons. While Netrin is an attractant cue in Drosophila axon guidance (Hiramoto and Hiromi, 2006;Brankatschk and Dickson, 2006), the role of its zebrafish ortholog is not completely resolved, even if a diffuse ntn1b expression within the somite is thought to promote ventral elongation of the CaP motor axon (Hale et al., 2011). The ventral restriction of uncx4.1 and ntn1b expression might involve a mechanism comprising the release of positional signals that contribute to the restriction of the CaP axon pathways. This may occur either by attracting CaP axons by diffusion of chemoattractants across intersomitic boundary epithelia, like in Drosophila, or repelling them within each somite through long-range cues (Mitchell et al., 1996). The CaP axon phenotype induced by uncx4.1 mRNA injection is similar to the effects of ectopically expressed netrins in other systems (Drosophila) (Mitchell et al., 1996). We speculate that the zebrafish Uncx4.1 activity in a particular subset of myotomal cells might serve a dual function by interacting with cell-cycle genes in controlling cell divisions during myoblast differentiation, and by activating or maintaining ntn1b expression for proper axonal elongation (Fig. 9).

Conflicts of interest
The authors declare that they have no conflict of interest.

Acknowledgments
We thank Rohan Mak (King's College) and Claire Williams (University College London) for technical help, and Chyenne Yeager (Wyss Institute) and Stefano Bellezza for English revision. This work was supported by a MIUR FIRB grant (RBFR12QW4I), a MIUR PON grant (a3_00239), and a PhD fellowship of the University of Geneva to PS, and by funding from the Wellcome Trust to SW. Funding sources had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. We dedicate this paper to the memory of Nigel Holder who died during the early course of the work.

Author contributions
VN, AED, GF, UC, AG, SM, FL, RDP, IP, RM and MF led the investigation and acquired the data. PS, AD, FA, TK, FR, LMC, DD and SW contributed to conceptualization, resources, data analysis and critical revision of the initial draft. UC, VN, GF and PS led the figure and table preparation. PS supervised the project, analyzed the data and drafted the manuscript, further completed by all authors. All authors have read and approved the manuscript.