Joshua F Coulcher

Agnès Roure

Rafath Chowdhury

Méryl Robert

Laury Lescat

Aurélie Bouin

Juliana Carvajal Cadavid

Hiroki Nishida

Sébastien Darras
email: sebastien.darras@obs-banyuls.fr

Ciinte Cagcgtgtccgtttgtttccttgttaatagaacttaattacagggtgtctgttgctatga

Cisavi Caccgtatcggttcgtttcctagttaatagaacataattacagggtggtgtgcgcaatgc

Ciinte Tgacaagataagatggcgggcgcttctcatctggcttgtttggcaacaattgatcgatag

Cisavi Taacaatacaaggt----Ggtctctctcatctactgtgtgtcgcaacaattgatcgatga

Ciinte Ccaaatcgcattatgagtcataactaaaattcgaatgcgggtggt-Gaattcgaacaaaa

Cisavi Cctaatcgtattataacttataattaaaactcgattacgacgaatggaaattgaaaaaaa

Tcatttttatgccataaataatcataaaatggctttcttaa

Ciinte Acgagtaattacggaaaaattatgacatcgtttaaatttcctacaactgtttacgtcgta

Cisavi Gcgagtaattacctcaaaattatggcaccgtttaaattcatttaaactgtttatgtcaca

Ciinte Agcgtctcattcattgtgggcgttta

Cisavi Gtaatgtacagcaagaaagctctttg

Conservation of peripheral nervous system formation mechanisms in divergent ascidian

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Abstract.

Ascidians with very similar embryos but highly divergent genomes are thought to have undergone extensive developmental system drift. We compared, in four species (Ciona and Phallusia for Phlebobranchia, Molgula and Halocynthia for Stolidobranchia), gene expression and gene regulation for a network of six transcription factors regulating peripheral nervous system (PNS) formation in Ciona.

All genes, but one in Molgula, were expressed in the PNS with some differences correlating with phylogenetic distance. Cross-species transgenesis indicated strong levels of conservation, except in Molgula, in gene regulation despite lack of sequence conservation of the enhancers. Developmental system drift in ascidians is thus higher for gene regulation than for gene expression; and is impacted not only by phylogenetic distance, but also in a clade-specific manner and unevenly within a network. Finally, considering that Molgula is divergent in our analyses, this suggests deep conservation of developmental mechanisms in ascidians after 390 My of separate evolution.

Introduction.

The formation of an animal during embryonic development is controlled by the exquisitely precise deployment of regulator and differentiation genes in space and time. This coordinated expression of developmental genes is controlled through gene regulatory networks (GRNs). It has been proposed that these networks are encoded in the genome and control body plan, organs and cell type formation [START_REF] Levine | Gene regulatory networks for development[END_REF]. It is thought that, in the course of evolution, essential GRNs or sub-modules have been conserved and control the formation of homologous structures, and that rewiring of the GRNs explains acquisition of novelties and morphological diversification [START_REF] Davidson | Gene regulatory networks and the evolution of animal body plans[END_REF].

Understanding control of gene expression is central to comparative developmental approaches. Hence, specific domains of the non-coding genome called cis-regulatory modules (CRMs) that serve as docking platforms for transcription factors (TFs) [START_REF] Buffry | Chapter Four -The Functionality and Evolution of Eukaryotic Transcriptional Enhancers[END_REF][START_REF] Rebeiz | Enhancer evolution and the origins of morphological novelty[END_REF] have been deeply studied. It is generally considered that during evolution and species diversification the key functional role of CRMs leads to their persistence and indirectly contributes to maintaining genes together (synteny) and to the existence of conserved non-coding DNA. Indeed, the presence of TF binding sites (TFBS) in the CRMs for genes with conserved expression and regulation defines regions of conserved sequence; and searching for conservation in non-coding DNA is often used to identify CRMs (phylogenetic footprint: defined as DNA sequence conservation between related species following alignment using tools such as lastz or lagan [START_REF] Brudno | LAGAN and Multi-LAGAN: Efficient Tools for Large-Scale Multiple Alignment of Genomic DNA[END_REF][START_REF] Schwartz | Human-Mouse Alignments with BLASTZ[END_REF]). Such conserved CRMs have been identified in closely related species but also to a fewer extent for very distantly related species (ultraconserved elements); and in many cases gene regulation relies on the usage of a similar combination of TFBS.

By contrast, a number of comparative studies performed in several group of animals (chordates, insects, nematodes…) have revealed that development is flexible and that formation of homologous characters may rely on different mechanisms, a process known as developmental system drift (DSD) [START_REF] Buffry | Chapter Four -The Functionality and Evolution of Eukaryotic Transcriptional Enhancers[END_REF][START_REF] Arnold | Quantitative genome-wide enhancer activity maps for five Drosophila species show functional enhancer conservation and turnover during cis-regulatory evolution[END_REF][START_REF] Verster | Comparative RNAi Screens in C. elegans and C. briggsae Reveal the Impact of Developmental System Drift on Gene Function[END_REF][START_REF] True | Developmental system drift and flexibility in evolutionary trajectories[END_REF][START_REF] Haag | The Same but Different: Worms Reveal the Pervasiveness of Developmental System Drift[END_REF][START_REF] Bradley | Binding Site Turnover Produces Pervasive Quantitative Changes in Transcription Factor Binding between Closely Related Drosophila Species[END_REF][START_REF] José-Edwards | Brachyury, Foxa2 and the cis-Regulatory Origins of the Notochord[END_REF][START_REF] Lusk | Evolutionary Mirages: Selection on Binding Site Composition Creates the Illusion of Conserved Grammars in Drosophila Enhancers[END_REF][START_REF] Roure | An Otx/Nodal Regulatory Signature for Posterior Neural Development in Ascidians. Desplan C[END_REF]. In some cases, key GRN nodes have changed (for example the role of Snail/Slug genes in vertebrate neural crest [START_REF] Nieto | A snail tale and the chicken embryo[END_REF]). Focusing on GRN edgesgene expression regulationvarious situations have been described, and conservation of CRM sequence at the DNA level does not necessarily correlate with conservation of activity. First, conservation of DNA sequence across species, does not imply a cis-regulatory activity (for example, ultraconserved elements do not necessarily function as CRMs or may be dispensable for gene expression) or an identical regulatory logic (conserved activity may not involve the same TFBS; conserved CRMs may control different expression patterns depending on the species). On the contrary, DNA sequence conservation is not a necessary feature of CRMs. First, bona fide CRMs may not be conserved, even between closely related species. Second, orthologous CRMs with conserved activity harboring similar combinations of TFBS exist despite an absence of sequence conservation because of extensive turnover of TFBS.

Ascidians are marine invertebrates that are particularly interesting for investigating the evolution of gene regulation during embryonic development. They belong to the tunicate phylum, and together with vertebrates and cephalochordates (amphioxus) they constitute the chordate super-phylum [START_REF] Lemaire | Evolutionary crossroads in developmental biology: the tunicates[END_REF][START_REF] Satoh | Developmental biology of ascidians[END_REF][START_REF] Satoh | Chordate evolution and the three-phylum system[END_REF]. These animals share a body plan characterized by a notochord and a dorsal neural tube during embryonic life.

Ascidians, however, took a significantly different evolutionary path from other chordates resulting in divergent morphological, embryological and genomic features.

The development of these animals is fast (hatching of the larva after 18hrs at 18°C in Ciona robusta [START_REF] Hotta | A webbased interactive developmental table for the ascidian Ciona intestinalis , including 3D real-image embryo reconstructions: I. From fertilized egg to hatching larva[END_REF]) and stereotyped with very few cells (around 100 cells in gastrulae and 2500 cells in larvae [START_REF] Nishida | Cell Division Pattern during Gastrulation of the Ascidian, Halocynthia roretzi. (cell division pattern/gastrulation/neurulation/ascidian embryo)[END_REF][START_REF] Yamada | Distinct parameters are involved in controlling the number of rounds of cell division in each tissue during ascidian embryogenesis[END_REF]), and ascidian genomes have undergone compaction and extensive rearrangements when compared to vertebrates [START_REF] Dehal | The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins[END_REF][START_REF] Brozovic | ANISEED 2017: extending the integrated ascidian database to the exploration and evolutionary comparison of genome-scale datasets[END_REF]. In addition, ascidians have extensively diversified (around 3,000 species) and their genomes have been reshuffled to the point that there is very little DNA conservation outside of the coding parts of the genome (except for closely related species such as the ones from the same genus) [START_REF] Shenkar | Global Diversity of Ascidiacea[END_REF][START_REF] Dardaillon | ANISEED 2019: 4D exploration of genetic data for an extended range of tunicates[END_REF]). Yet, their embryos are virtually identical. This paradoxical situation represents an excellent case to study the evolution and diversification of developmental mechanisms, CRMs and DSD [START_REF] José-Edwards | Brachyury, Foxa2 and the cis-Regulatory Origins of the Notochord[END_REF][START_REF] Roure | An Otx/Nodal Regulatory Signature for Posterior Neural Development in Ascidians. Desplan C[END_REF][START_REF] Stolfi | Divergent mechanisms regulate conserved cardiopharyngeal development and gene expression in distantly related ascidians[END_REF][START_REF] Madgwick | Evolution of embryonic cis-regulatory landscapes between divergent Phallusia and Ciona ascidians[END_REF][START_REF] Colgan | Variable levels of drift in tunicate cardiopharyngeal gene regulatory elements[END_REF][START_REF] Takahashi | Evolutionary alterations of the minimal promoter for notochord-specific Brachyury expression in ascidian embryos[END_REF][START_REF] Hudson | Divergent mechanisms specify chordate motoneurons: evidence from ascidians[END_REF][START_REF] Racioppi | Evolutionary loss of melanogenesis in the tunicate Molgula occulta[END_REF][START_REF] Johnson | Noncoding regulatory sequences of Ciona exhibit strong correspondence between evolutionary constraint and functional importance[END_REF][START_REF] Brown | Functional architecture and evolution of transcriptional elements that drive gene coexpression[END_REF][START_REF] Oda-Ishii | Making very similar embryos with divergent genomes: conservation of regulatory mechanisms of Otx between the ascidians Halocynthia roretzi and Ciona intestinalis[END_REF].

Ascidians are simple tractable organisms and are excellent models for functional genomics, in particular with plasmid DNA electroporation that allows the easy generation of thousands of transient transgenic embryos. Moreover, several species at various phylogenetic distances with sequenced and annotated genome are amenable to experimentation [START_REF] Dardaillon | ANISEED 2019: 4D exploration of genetic data for an extended range of tunicates[END_REF][START_REF] Kourakis | An organismal perspective on C. intestinalis development, origins and diversification[END_REF].

Several in details developmental studies have already been performed in ascidians and the comparative conclusions are contrasted (reviewed in [START_REF] Lemaire | Evolutionary crossroads in developmental biology: the tunicates[END_REF][START_REF] Kourakis | An organismal perspective on C. intestinalis development, origins and diversification[END_REF][START_REF] Hudson | Similarity and diversity in mechanisms of muscle fate induction between ascidian species. Biology of the cell / under the auspices of the European Cell Biology Organization[END_REF][START_REF] Lemaire | Ascidians and the plasticity of the chordate developmental program[END_REF][START_REF] Lemaire | Developmental biology. How many ways to make a chordate? Science[END_REF][START_REF] Satoh | Developmental genomics of ascidians[END_REF][START_REF] Razy-Krajka | Regulation and evolution of muscle development in tunicates[END_REF]). First, comparisons with vertebrates suggest that the 550 My of separate evolution has led to significant differences in the early specification of major chordate tissues such as the notochord or the central nervous system. But deep similarities have been uncovered in the GRN controlling heart or placode formation for example. Second, comparisons between distantly related ascidian species suggest that while the embryological and cellular processes are highly similar, the developmental regulators (TFs and signaling molecules) may change. Third, while conservation of developmental gene expression has been reported even at large phylogenetic distance, the mechanisms for such conservation relies on shared regulatory mechanism (i.e. conserved but shuffled TFBS in possibly non-alignable CRMs), or by contrast on different mechanisms (extensive DSD).

Comparative studies within ascidians are largely based on data from the most studied species Ciona intestinalis that have been compared with equivalent results obtained from another species belonging to a different family. Importantly, the estimated divergence times with C. intestinalis are considerable [START_REF] Delsuc | A phylogenomic framework and timescale for comparative studies of tunicates[END_REF]: 110 My for Ciona savignyi, 275 My for Phallusia mammillata or Corella inflata and 390 My for Halocynthia roretzi or Molgula species.

Here we have tried to expand the phylogenetic range by comparing gene expression regulation in several ascidian species, mainly C. intestinalis, P. mammillata and Molgula appendiculata. We have studied a subset of the GRN controlling caudal peripheral nervous system (PNS) formation. Caudal PNS formation is well understood in Ciona intestinalis both at the level of the developmental and cellular processes, and at the level of the molecular regulators [START_REF] Roure | An Otx/Nodal Regulatory Signature for Posterior Neural Development in Ascidians. Desplan C[END_REF][START_REF] Pasini | Formation of the Ascidian Epidermal Sensory Neurons: Insights into the Origin of the Chordate Peripheral Nervous System[END_REF][START_REF] Pasini | Antagonizing retinoic acid and FGF/MAPK pathways control posterior body patterning in the invertebrate chordate Ciona intestinalis[END_REF][START_REF] Roure | Msxb is a core component of the genetic circuitry specifying the dorsal and ventral neurogenic midlines in the ascidian embryo[END_REF][START_REF] Feinberg | Antero-posterior ectoderm patterning by canonical Wnt signaling during ascidian development[END_REF][START_REF] Waki | Genetic pathways for differentiation of the peripheral nervous system in ascidians[END_REF][START_REF] Chen | miR-124 function during Ciona intestinalis neuronal development includes extensive interaction with the Notch signaling pathway[END_REF][START_REF] Tang | Transcriptional regulation of the peripheral nervous system in Ciona intestinalis[END_REF][START_REF] Horie | Glutamatergic networks in the Ciona intestinalis larva[END_REF][START_REF] Candiani | Ci-POU-IV expression identifies PNS neurons in embryos and larvae of the ascidian Ciona intestinalis[END_REF][START_REF] Torrence | Nervous system of ascidian larvae: caudal primary sensory neurons[END_REF]. First, dorsal and ventral epidermis midlines are induced as neurogenic territories respectively by Fgf and Bmp signals from the underlying endomesodermal cells. Then, the number of glutamatergic PNS neurons (caudal epidermal sensory neurons or CESNs) that form in these neurogenic midlines is controlled by the Notch pathway. Molecular markers including a number of developmental regulators have been described for these two key phases of caudal PNS formation. However, their functions and direct interactions have been determined for only a limited number (Figure 1-figure supplement 1A).

Focusing on seven midline TFs, we identified CRMs that are active in neurogenic midlines from both C. intestinalis and P. mammillata. Reciprocal cross-species transcriptional assays pointed to a strong conservation of the cis-regulatory logic between these species despite a lack of sequence conservation in these CRMs.

Conserved activity was also observed in two additional species that both belong to the Phlebobranchia order. However, conserved activity was observed for only a minority of CRMs for some nodes of this GRN in M. appendiculata that belongs to the Solidobranchia order. Surprisingly, CRMs isolated from another Solidobranchia, Halocynthia roretzi, did show remarkable activity in the P. mammillata midlines but not in M. appendiculata. Overall, our results suggest deep conservation of gene regulation in caudal PNS formation within ascidians and advocate for broad phylogenetic sampling in comparative studies.

Results.

Identification of midline CRMs in Ciona intestinalis.

As described above, caudal PNS formation is initiated by two inductive events: Fgf9/16/20 as the inducer of the dorsal neurogenic midline (DML) and Admp as the inducer of the ventral neurogenic midline (VML). While function and regulatory interactions have been determined for several genes, direct interactions and associated CRMs are unknown for the most part (Fig 1-supplement 1A) [START_REF] Roure | An Otx/Nodal Regulatory Signature for Posterior Neural Development in Ascidians. Desplan C[END_REF][START_REF] Pasini | Formation of the Ascidian Epidermal Sensory Neurons: Insights into the Origin of the Chordate Peripheral Nervous System[END_REF][START_REF] Roure | Msxb is a core component of the genetic circuitry specifying the dorsal and ventral neurogenic midlines in the ascidian embryo[END_REF][START_REF] Waki | Genetic pathways for differentiation of the peripheral nervous system in ascidians[END_REF][START_REF] Tang | Transcriptional regulation of the peripheral nervous system in Ciona intestinalis[END_REF][START_REF] Bertrand | Neural tissue in ascidian embryos is induced by FGF9/16/20, acting via a combination of maternal GATA and Ets transcription factors[END_REF]. Here, we have focused our work on seven TFs that are expressed in both ventral and dorsal caudal neurogenic midlines (VDML) and for which we have already described regulatory interactions that generated a provisional GRN [START_REF] Pasini | Formation of the Ascidian Epidermal Sensory Neurons: Insights into the Origin of the Chordate Peripheral Nervous System[END_REF][START_REF] Roure | Msxb is a core component of the genetic circuitry specifying the dorsal and ventral neurogenic midlines in the ascidian embryo[END_REF]. These genes areordered by their onset of expression in VDML - for a C2H2 Zn finger TF; and Tox for a HMG TF. We aimed at identifying regions of genomic DNA that behave as midline CRMs. Previous work in the ascidian community has shown that selecting a few kb in conserved non-coding DNA generally upstream of the gene of interest had high chance of identifying most, if not all, regulatory regions. We thus selected upstream regions of up to 7 kb containing phylogenetic footprints between C. robusta and C. savignyi that we placed upstream of the minimal promoter of the Ciinte.Fog gene and LacZ as a reporter, and tested in vivo before further reduction in size. After our work was performed, open chromatin regions from ATAC-seq data were identified from whole embryos up to neurula stages [START_REF] Madgwick | Evolution of embryonic cis-regulatory landscapes between divergent Phallusia and Ciona ascidians[END_REF]; and it turned out that most of the midline CRMs that we identified correspond to conserved regions with enrichment in ATAC-seq (Figures 123, Supplementary file 1 and 2). Tissue-specific ATAC-seq data should thus have a high predictive power and limit the time-consuming promoter bashing approach in future experiments seeking for active CRMs.

Msx

Uncoupling of dorsal and ventral activities for the early genes.

Msx.

Msx is expressed in DML precursors from the 64-cell stage while it is expressed in the VML from the gastrula/neurula stages [START_REF] Roure | Msxb is a core component of the genetic circuitry specifying the dorsal and ventral neurogenic midlines in the ascidian embryo[END_REF]. We have previously described a Nodal/Otx-regulated CRM located immediately upstream of the gene responsible for the early expression in DML precursors (Ci-msxb-b6.5 line in Figure 1A) [START_REF] Roure | An Otx/Nodal Regulatory Signature for Posterior Neural Development in Ascidians. Desplan C[END_REF]. Here, we show that the entire upstream intergenic region is active early in the DML (Figure 1B) but also later in the VML (Figure 1C). A distal fragment, up1, adjacent to the early CRM was active in both VML and DML at tailbud stages but inactive at gastrula/neurula stages (Figure 1D-F). These results show that the seemingly continuous expression of Msx in the DML is in fact under the control of two separate CRMs that are likely regulated differently (see below). We next generated various deletions of this VDML CRM (Figure 1F and Supplementary file 2). We could observe VDML activity for most regions, with generally a decrease of strength when the size of the fragment was reduced, or a lack of activity. The smallest fragment active in VDML, up10, was 291 bp long. Interestingly, two overlapping regions, up2 and up11, were active only in the DML. Consequently, even if we did not identify a CRM specific for the VML, this deletion analysis suggests that ventral and dorsal expressions of Msx are uncoupled, a conclusion in agreement with the proposed role of Msx as an upstream regulator of the neurogenic GRN and an integrator of dorsal and ventral inducing cues [START_REF] Roure | Msxb is a core component of the genetic circuitry specifying the dorsal and ventral neurogenic midlines in the ascidian embryo[END_REF]. This scenario is further supported by the presence of conserved

Ascl.b.

While the upstream intergenic region (0.8 kb) was not active (Supplementary file 2), the 0.7 kb region immediately downstream of the gene, do1, was robustly active in VDML (Figure 2 and Supplementary file 2). Fragments of this region were either not active, active in VDML or mainly active in the DML. Consequently, similarly to Msx, the VML and DML expression of Ascl.b appear to be distinctly regulated. While we identified putative sites for dorsal and ventral factors in the do2 fragment (Fig 2 -supplement 1), they are not sufficient to explain VDML activity. This suggests that Ascl.b expression relies on additional unidentified factors.

Pan-midline CRMs for late midline genes.

For the five other TF genes, we could also isolate genomic regions active in VDML, except for Bhlhtun1 for which the 3 tested regions were active in endogenous expression territories (palps, notochord…) but not in VDML (Supplementary file 2 and 3).

Klf1/2/4.

A 6.6 kb region, up1, immediately upstream of the gene was strongly active in VDML (Figure 3A and Figure 3-figure supplement 1). Various deletions were generated; most were active in VDML but with a lower efficiency (Figure 3 We could identify a 326 bp fragment, up7, which was robustly active in VDML (Figure 3D). It is located 0.7 kb upstream of Nkxtun3 and corresponds to a conserved region with an ATAC-seq enrichment detected from late gastrula stages (Supplementary file 2). TFBS analysis suggested activation by the upstream factors Msx and Klf1/2/4 (Figure 3-figure supplement 2).

Tox.

Tox is mainly transcribed as two isoforms that vary in their transcription start sites and first exons (Figure 3-figure supplement 3 and Supplementary file 2) [START_REF] Roure | Msxb is a core component of the genetic circuitry specifying the dorsal and ventral neurogenic midlines in the ascidian embryo[END_REF]. We selected a 913 bp conserved region, int1, located upstream of one isoform and in the first intron of the other. This region is active in VDML (Figure 3E). Two shorter regions were generated: int1.1 (565 bp) that is more active (Figure 3F) and int1.2 (138 bp) that is less (Figure 3

Dlx.c.

A 4.8 kb region, A2, immediately upstream of the gene was strongly active in VDML (Figure 3G and Figure 3-figure supplement 4). Various deletions were generated; most were active in VDML but with a lower efficiency (Figure 3-figure supplement 4).

The region 'upstream' (604 bp), located 1.3 kb upstream of Dlx.c, contains two blocks with higher sequence conservation and ATAC-seq enrichment, and was robustly active (Figure 3H). up7 (206 bp) containing the proximal block was active in VDML albeit at a low level (Figure 3-figure supplement 4). It does not contain any site for the putative upstream VDML regulators (Msx, Ascl.b, Klf1/2/4 and Nkxtun3), suggesting the implication of unidentified factor(s).

Interestingly, for all these 4 TFs coding genes (Klf1/2/4, Nkxtun3, Tox and Dlx.c), starting from relatively large regions (up to 6.6 kb), we could reduce the size down to 200-300 bp and maintain VDML activity (Figure 3). Shortening the DNA regions was usually accompanied by a decrease in activity. However, all tested regions, despite some variability, had similar activity in both VML and DML; and we conclude that the expression of these genes is regulated by 'pan-midline' CRMs in agreement with their downstream positions in the PNS GRN [START_REF] Roure | Msxb is a core component of the genetic circuitry specifying the dorsal and ventral neurogenic midlines in the ascidian embryo[END_REF].

Overall, results from promoter bashing in C. intestinalis led to three main conclusions: genomic support for VDML expression has been identified for most midline TFs (and this will be used to probe gene regulation conservation in other species); Msx and Ascl.b have been confirmed as upstream regulators of the caudal PNS GRN since dorsal and ventral expression are differently regulated; and downstream genes are regulated similarly dorsally and ventrally.

Tail PNS specification appears to be conserved in Phallusia mammillata.

We next wondered whether caudal PNS was specified by similar mechanisms in Phallusia mammillata, an ascidian species that diverged from C. intestinalis around 275 My ago (41) (Figure 4). We have previously shown that the early induction of DML fate by Fgf/Nodal signals is conserved and leads to the expression of the early DML precursors markers Msx and Dlk (previously known as Delta2) [START_REF] Roure | An Otx/Nodal Regulatory Signature for Posterior Neural Development in Ascidians. Desplan C[END_REF].

Expression and regulation of caudal PNS genes.

We first determined by in situ hybridization the expression pattern of orthologous genes for the caudal PNS genes (for each gene, we could identify a single ortholog in the Phallusia genome). Importantly, we found that the seven neurogenic TFs, that are the focus of the present study, are expressed in VDML like in Ciona and with apparently the same temporal sequence (Figure 4B We next determined whether Bmp and Notch signaling pathways could regulate caudal PNS formation. Treatment of whole embryos with zebrafish recombinant Bmp4 protein from the 8-cell stage led to ectopic expression in the lateral tail epidermis of Msx (BSA control: 20/20 embryos in VDML, N=1; 150 ng/ml Bmp4: 27/27 embryos with ectopic expression, N=1), Klf1/2/4 (BSA control: 100% embryos in VDML, N=9, n=172; 150 ng/ml Bmp4: 100% embryos with ectopic expression, N=6, n=127; Figure 4M,O) and Pou4F (BSA control: 100% embryos in VDML, N=4, n=101; 150 ng/ml Bmp4: 94% embryos with ectopic expression, N=4, n=95; Figure 4R,S). Overexpression of the Ciona Bmp ligand Admp in the ectoderm by electroporation led also to ectopic Klf1/2/4 expression (pFog>Venus control: 100% embryos in VDML, N=4, n=75; pFog>Ciinte.Admp: 71% embryos with ectopic expression, N=1, n=14; Figure 4M,N). Bmp inhibition using the pharmacological inhibitor Dorsomorphin had variable effects depending on the experiment, but we observed, as in Ciona, repression of Klf1/2/4 expression in the VML (BSA control: 100% embryos in VDML, N=9, n=172; 20 μM Dorsomorphin: 5 to 70% embryos with VML repression with an average of 37%, N=9, n=200; Figure 4M,Q). Similarly overexpression of the secreted Bmp antagonist Noggin from Ciona led to VML repression of Klf1/2/4 expression (pFog>Venus control: 100% embryos in VDML, N=4, n=75; pFog>Ciinte.Noggin: 0 to 80% embryos with VML repression with an average of 29%, N=4, n=109; Figure 4M,P). Inhibition of Notch signaling with the pharmacological inhibitor DAPT led to a massive increase of the Pou4F positive cells in VDML; and when combined with Bmp4 treatment, the cells were found throughout the tail epidermis (BSA control: 100% embryos in VDML, N=4, n=101; 25 μM DAPT: 99% embryos with ectopic expression in VDML, N=3, n=94; 25 μM DAPT + 150 ng/ml Bmp4: 93% embryos with ectopic expression, N=4, n=118; Figure 4R,T,U).

Identification of midline CRMs.

To define VDML CRMs in P. mammillata, we undertook the same approach as the one performed in C. intestinalis but with a more limited generation of deletion constructs. We selected regions containing blocks of sequence conservation between P. mammillata and P. fumigata that we placed upstream of the minimal promoter for the Ciinte.Fog gene and LacZ as a reporter. Out of the 7 targeted genes, we were unable to isolate CRMs with VDML activity for Ascl.b and Bhlhtun1 (Supplementary file 4 and 5).

Msx.

Similarly to the situation in C. intestinalis, we have previously isolated Pm-msxb-b6.5 line, a CRM immediately upstream of Msx that is active early in the DML precursors (b6.5 lineage) [START_REF] Roure | An Otx/Nodal Regulatory Signature for Posterior Neural Development in Ascidians. Desplan C[END_REF]. The upstream intergenic region that contains this CRM was active at tailbud stages in VDML (Figure 5A and Figure 5-figure supplement 1), and allowed to define a distal CRM, LED, that was active in VDML and corresponds to a conserved region with a weak ATAC-seq signal (Figure 5-figure supplement 1 and A short conserved region immediately upstream of Tox, A (1093 bp), was strongly active in VDML but also in tail muscle and mesenchyme (Figure 5D, Figure 5-figure supplement 3 and Supplementary file 5). Activity in muscle/mesenchyme segregated to a proximal fragment, A2 (533 bp), while VDML activity segregated to a distal fragment, A1 (657 bp), which could be reduced to 240 bp (A1.1) with maintaining a robust VDML activity. The small size of these fragments allowed TFBS analysis (Figure 5-figure supplement 3) that suggested a possible regulation by Ascl.b as for Ciinte.Tox (Figure 3-figure supplement 3).

Dlx.c.

Based on RNA-seq profiling, we estimated that the predicted gene model for Dlx.c was lacking its first exon located approximately 8 kb upstream (Supplementary file 5).

We thus isolated a 5.4 kb region containing 2 blocks of conservation, A, which was strongly active in VDML (Figure 5E and

Conserved midline PNS gene regulation in Phlebobranchia.

Here we directly investigated whether the expression of caudal neurogenic midlines genes was regulated similarly between C. intestinalis and P. mammillata. To this end, we performed bi-directional 'enhancer swaps': we tested in P. mammillata embryos the activity of C. intestinalis VDML CRMs and vice versa. Strikingly, all the regions that we have tested were active in VDML in over 40-50% of the embryos in the recipient species (Figure 6A

Minimal enhancers are not robust.

Above results suggest a strong conservation of the caudal PNS GRN at the level of gene expression, gene regulation and its genomic encoding (CRMs) in Phlebobranchia ascidians. However, potentially homologous CRMs (CRMs from orthologous genes, located at similar positions in their locus and with conserved VDML activity in swap experiments), do not show significant DNA sequence conservation at large distance (i.e. between C. intestinalis and P. mammillata) (Supplementary file 2 and 5). There are previous examples in ascidians that clearly show that such conserved activity relies on conserved upstream regulators but that extensive binding site turnover explains the lack of sequence conservation [START_REF] Roure | An Otx/Nodal Regulatory Signature for Posterior Neural Development in Ascidians. Desplan C[END_REF][START_REF] Madgwick | Evolution of embryonic cis-regulatory landscapes between divergent Phallusia and Ciona ascidians[END_REF][START_REF] Colgan | Variable levels of drift in tunicate cardiopharyngeal gene regulatory elements[END_REF]. We thus reasoned that the CRMs with conserved activity that we have isolated from C. intestinalis and P. mammillata could help in identifying conserved TFBS and shared TF regulating CRM activity. We also postulated that this approach would be effective with CRMs of small size. By applying a strict conservative approach on known putative regulators, we could hypothesize that Msx expression in the VDML from gastrula/neurula stages is directly regulated by Bmp signaling triggered by Admp in the VML, and by Msx itself in the DML (Figure 1 We next focused on the proximal Ci-msxb-b6.5 line CRM that regulates early Ciinte.Msx expression in DML precursors (Figure 1A) [START_REF] Roure | An Otx/Nodal Regulatory Signature for Posterior Neural Development in Ascidians. Desplan C[END_REF]. We have previously shown that its size could be reduced from 707 bp to 273 bp (Ci-msxb-B) while maintaining a robust activity (around 50% of embryos with DML activity) (Figure 7A). Surprisingly, when the various deletions of Ci-msxb-b6.5 were tested in P. mammillata, they were barely active (<10% for constructs of 400 bp or less) (Figure 7A). Since these different constructs still contained the Otx and Smad binding sites that are essential for Ci-msxb-B activity in C. intestinalis, we wondered whether Cimsxb-B was faithfully recapitulating Ciinte.Msx expression. To this end, we challenged Ci-msxb-B with known regulators of Ciinte.Msx expression (Figure 7B). We thus wondered whether other short CRMs that we have described here with VDML activity also behaved inappropriately when submitted to a genetic challenge.

We thus determined the activity of these CRMs when a midline TF was overexpressed and compared the effects with what we have already described for the endogenous expression of the associated gene [START_REF] Roure | Msxb is a core component of the genetic circuitry specifying the dorsal and ventral neurogenic midlines in the ascidian embryo[END_REF]. We were very surprised to realize to while some interactions were identical, a large part was different (Figure 7C). Also, two different CRMs for Ciinte.Nkxtun3 behaved differently.

We conclude from this section that short or minimal CRMs should be taken with cautious since they may not be robust to phylogenetic (enhancer swap) or genetic (overexpression) challenges.

Variable levels of divergence in Stolidobranchia.

Here we aimed at exploring whether the striking conservation of VDML GRN holds true outside Phlebobranchia. We first determined the expression patterns of several genes of this network during the development of two solitary Stolidobranchia species, Molgula appendiculata and Halocynthia roretzi (Figure 8, Figure 8-figure supplement 1-3). Both species are estimated to have diverged around 340 My ago, and 390 My ago with Phlebobranchia (Figure 8A) [START_REF] Delsuc | A phylogenomic framework and timescale for comparative studies of tunicates[END_REF]. Using transcriptomic data for M. appendiculata and both transcriptomic and genomic data for H. roretzi [START_REF] Dardaillon | ANISEED 2019: 4D exploration of genetic data for an extended range of tunicates[END_REF], we identified a single ortholog for each gene that we analyzed, except for Ascl.b for which two paralogs were found in M. appendiculata.

Gene expression in M. appendiculata.

For the orthologs of neurogenic midline genes, three had an expression very similar ). While Moappe.Dlk was expressed in mesoderm and central nervous system, we did not detect a specific expression in the VDML. Moappe.Celf3.a was expressed strongly in the central nervous system as in Phlebobranchia, and also in isolated spots on surface ectoderm that likely correspond to ectodermal sensory neurons. In particular, Moappe.Celf3.a was detected in two to five spots of the posterior VDML, suggesting that the number of CESNs in this species is small.

Moreover, this caudal restriction of expression is in agreement with the expression of

Moappe.Nkxtun3, Moappe.Ascl.bβ and Moappe.Tox.

Gene expression in H. roretzi.

Among the orthologs of neurogenic genes, Harore.Dlx.c pattern had already been described as expressed in the VDML (53) (Figure 8M Overall, comparison of gene expression shows striking conservation in patterns but also in the temporal order despite almost 400 My of divergence between Stolidobranchia and Phlebobranchia. However, the degree of conservation is weaker when considering M. appendiculata.

CRM swap.

We next wondered whether conservation could extend to CRM activity. We thus tested various CRMs isolated from C. intestinalis and P. mammillata in M. appendiculata that is amenable to electroporation. A first qualitative analysis revealed that CRMs for several midline genes were found active in M. appendiculata VDML: 9A), Ascl.b (Figure 9B), Nkxtun3 (Figure 9D) and Dlx.c (Figure 9F). In other cases, CRMs were active but not in VDML: anterior ectoderm and central nervous system for Ciinte.Klf1/2/4-up1 (Figure 9C), or tail muscle for Phmamm.Tox-A (Figure 9E). However, when the strength of the activity was considered (measured indirectly as the percentage of stained embryo in VDML), the situation appeared quite different. VDML activity was seldom observed for CRMs from Msx and Ascl.b (Figure 9-figure supplement 1). These were only large CRMs for Nkxtun3 and Dlx.c that were robustly active in M. appendiculata VDML. The overall low levels of activity for the tested CRMs could be explained by a non-optimal electroporation procedure.

Msx (Figure
While this cannot completely be ruled out, electroporation of the regulatory sequences for the ubiquitous gene Cirobu.Ef1α (45) led to strong expression of the LacZ reporter gene (89% of stained embryos, n=74 in 2 independent experiments) and suggested a rather efficient procedure. Interestingly, for both Nkxtun3 and Dlx.c, CRMs from both C. intestinalis and P. mammillata were active in Molgula VDML, supporting the specificity of the results. This further suggests that within the 6-genes network that we have explored, only these two genes have retained conserved regulation.

To further explore whether the global lack of conservation that we have observed in M. appendiculata could be a generality in Stolidobranchia, we have isolated genomic upstream regions for Msx, Nkxtun3 and Tox from H. roretzi (Figure 9-figure supplement 2 and Supplementary file 7). The 3 regions were poorly active in M. appendiculata embryos except Harore.Nkxtun3-B that was active in anterior sensory vesicle in up to 29% of the embryos (Figure 9I). Only Harore.Msx-A had a striking VDML pattern but in a minority of embryos (Figure 9G). These results could simply be that these regions do not behave as VDML CRMs since we have not determined their activity in H. roretzi. However, in P. mammillata, while Harore.Nkxtun3-B was weakly active in VDML with additional staining in lateral tail epidermis (Figure 9J and

Regulation of midline gene expression in Ciona intestinalis.

We have reported the identification of midline CRMs for 6 out of the 7 genes that we have tackled. Our approach was simply based on isolating conserved regions (with C. savignyi) close to the gene of interest. Similarly to numerous reports, active CRMs were found mostly within the first few kilobases immediately upstream of the gene (a single region was located downstream). Interestingly, the largest CRMs that we have tested contain activity for several expression domains where the gene is expressed and do not usually display ectopic activity. We further reduced the size of these CRMs through classical 'promoter bashing' guided by sequence conservation.

Activity in different domains usually partitioned during this deletion process while we have not documented this in detail (for example for Dlx.c, palp expression in the large region Ciinte.Dlx.c-A2 is lost from the small region Ciinte.Dlx.c-upstream; Figure 3G While our approach was not exhaustive, important conclusions could be drawn. First, the seemingly continuous expression of Msx in the b6.5 lineage and its derivative, the DML, is actually under the control of two separate CRM: an early proximal element active from the 64-cell stage ( 14) and a late distal element active from gastrula stages (Figure 1). TFBS analysis using our current understanding of Msx regulation (Figure 1

High conservation of PNS formation in Phlebobranchia.

Expression patterns, overexpression experiments and CRMs identification in P. mammillata showed high similarities with C. intestinalis (Figure 4, 5 and 10). This was further demonstrated by CRM swap experiments (Figure 6 and 10) where midline CRMs from C. intestinalis were active in P. mammillata midlines and vice versa, and also in two additional Phlebobranchia species (Figure 6). These observations suggest that orthologous genes are regulated similarly and that CRMs are controlled using the same regulatory logic. Since non-coding DNA does not show sequence conservation, for example between C. intestinalis and P. mammillata CRMs that we identified (Supplementary file 2 and 5), it is difficult to define homologous CRMs. This is likely explained by extensive turnover of the binding sites for upstream transcription factors, a situation that we have previously described for the regulation of the early Msx CRMs by Otx and Nodal [START_REF] Roure | An Otx/Nodal Regulatory Signature for Posterior Neural Development in Ascidians. Desplan C[END_REF]. The newly identified Msx CRM from A. mentula actually contains a region dense in Otx and Smad binding sites like their counterparts in Ciona and Phallusia (not shown). This phenomenon has been described for other CRMs between C. intestinalis and other species and outside ascidians [START_REF] Buffry | Chapter Four -The Functionality and Evolution of Eukaryotic Transcriptional Enhancers[END_REF][START_REF] Colgan | Variable levels of drift in tunicate cardiopharyngeal gene regulatory elements[END_REF][START_REF] Oda-Ishii | Making very similar embryos with divergent genomes: conservation of regulatory mechanisms of Otx between the ascidians Halocynthia roretzi and Ciona intestinalis[END_REF].

Given the modular nature of cis-regulation and the increased recognition of the existence of 'redundant', 'shadow' or distributed enhancers [START_REF] Barolo | Shadow enhancers: Frequently asked questions about distributed cis-regulatory information and enhancer redundancy[END_REF][START_REF] Cannavò | Shadow Enhancers Are Pervasive Features of Developmental Regulatory Networks[END_REF], namely multiple CRMs with seemingly similar activity for a given gene, caution has to be taken for pairwise comparisons in TFBS composition of genomic pieces originating from different species. We could nevertheless apply the above approach for two other CRMs. In both Ciona and Phallusia, the late Msx CRMs with VDML activity are distal and share binding sites for Msx and Bmp regulation (SBE and BRE) (Figure 1 We thus aimed at using such a strategy using candidate orthologous minimal midline CRMs to define shared motifs and identify trans-acting factors. However, the 'swap' and overexpression experiments described in Figure 7 refrained us from pursuing since such CRMs were not robust to phylogenetic and genetic perturbations.

Whether these observations are general to ascidian minimal CRMs remains to be explored. A possible interpretation could be linked to the modular nature of cisregulation. The largest CRMs that we tested are likely to contain most, if not all, the regulatory information for VDML expression. By contrast, shorter elements contain only a subset of the regulatory information, and would consequently not behave identically to the gene following genetic challenge. The fact, that such short elements may not be active when placed in another species while larger regions are, suggests that drift has occurred at the level of individual modules making them silent. Independently of our current understanding of this phenomenom, reciprocal in vivo transcriptional assays between Ciona and Phallusia offer a direct way to identify and determine the properties of the DNA elements that confer robustness.

Global conservation of PNS formation in ascidians?

In situ hybridization in two members of the Stolidobranchia revealed strong conservation in gene expression. However, while the patterns were virtually identical between Ciona and Phallusia, we could detect several differences (Figure 8 When focusing on gene regulation, we detected again stronger conservation within Phlebobranchia than between Phlebobranchia and Stolidobranchia (Figure 10). This is particularly true when considering large CRMs of several kilobases that have better chance of capturing the widest regulatory information. This difference between ascidian orders could be the result of increased drift because of larger evolutionary distance and/or because of discrete changes linked to taxonomical level change. We were nevertheless surprised to see that CRMs from Halocynthia were active in midlines when tested in Phallusia but not in Molgula (Figure 9 and 10). While we have obtained these results on a limited number of CRMs and while we have not tested CRMs from M. appendiculata whose genome has not been sequenced, we believe that, taking expression patterns into account, M. appendiculata is the divergent species among all species that we have used (Figure 10). Our hypothesis is in agreement with previous CRM swap experiments between Ciona and other Molgula species [START_REF] Stolfi | Divergent mechanisms regulate conserved cardiopharyngeal development and gene expression in distantly related ascidians[END_REF], with the facts that Molgula is a fast evolving genus within ascidians [START_REF] Delsuc | A phylogenomic framework and timescale for comparative studies of tunicates[END_REF][START_REF] Alié | Convergent Acquisition of Nonembryonic Development in Styelid Ascidians[END_REF] and that Molgulids is a family where loss of chordate body plan has occurred several times independently (tail-less larvae) [START_REF] Huber | The evolution of anural larvae in molgulid ascidians[END_REF]. Surprisingly, we did not detect obvious correlations between points of conservation in gene regulation and gene expression or GRN structure. Nkxtun3 and Dlx.c are the two genes whose CRMs are active in Molgula midlines (Figure 9 and Figure 9-figure supplement 1). They are not the genes with the best conserved expression; and while Dlx.c is in a downstream position of the network, Nkxtun3 has an intermediate position [START_REF] Roure | Msxb is a core component of the genetic circuitry specifying the dorsal and ventral neurogenic midlines in the ascidian embryo[END_REF]. Interestingly, in M. appendiculata Nkxtun3 is found only in posterior VDML and Dlx.c is not expressed in the VDML, but CRMs from both Ciona and Phallusia were active throughout M. appendiculata VDML. Consequently, it is likely that upstream factors are likely expressed throughout the VDML and that changes in the cis-regulatory landscapes have occurred for both genes in M. appendiculata.

Similarly, a CRM from Halocynthia Tox (expressed in a spotted manner) was active throughout the VDML in Phallusia.

Given the phylogenetic positions of the species (Figure 8A) and the divergent behavior of Molgula (see above), a parsimonious analysis of our results (summarized in Figure 10) implies that PNS gene expression and regulation are globally conserved in ascidians despite extensive genomic divergence in the course of almost 400 My of evolution. This is a similar time window that separates mammals from teleosts [START_REF] Delsuc | A phylogenomic framework and timescale for comparative studies of tunicates[END_REF], and several developmental processes including CRM activity have been shown conserved between mouse and zebrafish for example, or even between more divergent vertebrate species (zebrafish and lamprey) [START_REF] Hockman | A genome-wide assessment of the ancestral neural crest gene regulatory network[END_REF][START_REF] Fisher | Conservation of RET Regulatory Function from Human to Zebrafish Without Sequence Similarity[END_REF][START_REF] Gehrke | Deep conservation of wrist and digit enhancers in fish[END_REF][START_REF] Mcgaughey | Metrics of sequence constraint overlook regulatory sequences in an exhaustive analysis at phox2b[END_REF]. While some of the cited examples refer to CRM conserved activity despite lack of sequence conservation, in vertebrates CRMs are usually associated with sequence conservation. In ascidians, the situation is the opposite with an absence of noncoding DNA conservation and lack of synteny.

We would like to conclude by stressing two operational points for comparative studies in ascidians that may be relevant more broadly. The CRMs are interesting tools to probe gene regulation especially during swap experiments in species with efficient transgenesis. However, the lack of fidelity of small CRMs that we have uncovered may blur comparative conclusions. Finally, the choice of species for comparisons is also essential. While divergent species are highly informative to understand evolution at work, they may lead to inaccurate conclusions at a broader level. Although the established model species Ciona intestinalis and Halocynthia roretzi appear to be relevant species to instruct the status of ancestral ascidians, we would advocate for broad taxonomic sampling in functional comparative studies.

Materials and Methods.

Embryo obtention and manipulation.

Adults from Ciona intestinalis (formerly referred to Ciona intestinalis type B (68)) and

Ascidia mentula were provided by the Centre de Ressources Biologiques Marines in Roscoff (EMBRC-France). Phallusia fumigata and Molgula appendiculata were provided by the Centre de Ressources Biologiques Marines in Banyuls-sur-mer (EMBRC-France). Phallusia mammillata were collected during trawling by professional fishermen in the Banyuls-sur-mer area. Gametes were collected from the gonoducts, except for M. appendiculata where gonads dissociation released sperm and immature eggs that spontaneously matured in sea water within 20-30 min.

Egg dechorionation was performed before fertilization for P. fumigata and A. mentula as previously described for P. mammillata [START_REF] Roure | An Otx/Nodal Regulatory Signature for Posterior Neural Development in Ascidians. Desplan C[END_REF], and after fertilization for M. appendiculata as described for C. intestinalis [START_REF] Mita-Miyazawa | Histospecific acetylcholinesterase development in the presumptive muscle cells isolated from 16-cell-stage ascidian embryos with respect to the number of DNA replications[END_REF]. 50 μg of plasmid DNA was electroporated as described previously [START_REF] Feinberg | Antero-posterior ectoderm patterning by canonical Wnt signaling during ascidian development[END_REF] through a single 32 ms electrical pulse with the following voltage: 20V for M. appendiculata, 25V for C. intestinalis and 37V for P. mammillata, P. fumigata and A. mentula. P. mammillata embryos were treated with 150 ng/ml recombinant zebrafish Bmp4 protein (1128-BM, R&D Systems, stock solution in HCl 4mM + BSA 0,1%) complemented with 0.1% BSA from the 8-cell stage, with 20 μM of the Bmp receptor inhibitor Dorsomorphin (S7306, Euromedex, stock solution in water) from early gastrula stages or with 25 μM of the γ-secretase inhibitor DAPT (D5942, Sigma-Aldrich, stock solution in DMSO) from early neurula stages. Control embryos were grown in seawater containing 0.1% BSA and 0.5% DMSO. Whole mount in situ hybridization were performed as described [START_REF] Feinberg | Antero-posterior ectoderm patterning by canonical Wnt signaling during ascidian development[END_REF] with Dig-labeled probes synthesized from clones described in Supplementary file 8. These clones were selected from EST libraries or PCR-amplified based on genomic or/and transcriptomic data (Supplementary file 9) [START_REF] Dardaillon | ANISEED 2019: 4D exploration of genetic data for an extended range of tunicates[END_REF][START_REF] Kawashima | MAGEST: MAboya gene expression patterns and sequence tags[END_REF][START_REF] Kawashima | Update of MAGEST: Maboya Gene Expression patterns and Sequence Tags[END_REF]. Reference transcriptome for M. appendiculata was generated from RNA-seq data on mixed embryonic stages and will be described elsewhere. 10-15 control embros per stage were examined for normal expression patterns, while 40-60 embryos were analyzed in the case of perturbations.

In vivo transcriptional assay.

Genomic regions were PCR amplified and placed upstream of the minimal promoter of the Ciinte.Fog gene (KH2012:KH.C10.574) (except for 3 regions directly abutting the transcribed region of the gene: Ciinte.Nkxtun3-upstream, Ciinte.Ascl.b-upstream and Ciinte.Bhlhtun1-upstream) and the LacZ reporter gene using the Gateway technology (Invitrogen) as described previously [START_REF] Roure | A multicassette Gateway vector set for high throughput and comparative analyses in ciona and vertebrate embryos[END_REF]. Details for newly isolated genomic regions, primers, templates and vectors are available in Supplementary file 1. Details for already described regions (Figure 1 and Figure 5-figure supplement 1) can be found in [START_REF] Roure | An Otx/Nodal Regulatory Signature for Posterior Neural Development in Ascidians. Desplan C[END_REF]. In the case of Msx in A. mentula, for which no whole genome sequence is available, we reasoned that local synteny would be conserved given the phylogenetic relationships between Ascidia, Phallusia and Ciona genera (Figure 6A).

Since the gene upstream of Msx is orthologous in sequenced Ciona and Phallusia species (KH2012:KH.C2.808 in C. intestinalis; Phmamm.g00005894 in P. mammillata), we amplified the Msx upstream intergenic region using degenerate primers defined in the coding sequences of Msx and its upstream neighbor (Fwd: TTYGAYAARTAYCARTTYGA; Rev: TTYTCYTGRAAYTTRTTYTC). Sequencing of this amplicon led to designing new primers for PCR amplification and cloning (Supplementary file 1). The sequence of the amplified region, Asment.Msx-up, can be found in Supplementary file 9.

Following electroporation and development, embryos were fixed at the desired stage and stained for β-galactosidase activity using X-gal [START_REF] Roure | An Otx/Nodal Regulatory Signature for Posterior Neural Development in Ascidians. Desplan C[END_REF]. For each experiment, the number of embryos with β-galactosidase-positive cells in VDML was scored (a total of 30 to 300 embryos were counted per experiment). The number of experiments performed and the number of embryos examined is described in the text and figures.

Graphs in the figures represent average values, with error bars denoting the standard deviation.

In the case of concomitant overexpression of a TF in C. intestinalis (Figure 7), at least two independent experiments have been performed, but without scoring.

Constructs for TF overexpression have been previously described [START_REF] Roure | Msxb is a core component of the genetic circuitry specifying the dorsal and ventral neurogenic midlines in the ascidian embryo[END_REF].

Gene model identifiers.

The genes described in this study were named according to the nomenclature in the tunicate community [START_REF] Stolfi | Guidelines for the nomenclature of genetic elements in tunicate genomes[END_REF] and this may differ from previous publications. Homologous genes were identified using blast against genome/transcriptome and pre-computed orthologies when available from the Aniseed database [START_REF] Dardaillon | ANISEED 2019: 4D exploration of genetic data for an extended range of tunicates[END_REF]. Gene identifiers come from the following assemblies: C. robusta KH2012, P. mammillata MTP2014, P. fumigata MTP2014 and H. roretzi MTP2014 (Supplementary file 1). In some cases, gene or transcript models were absent or incorrect; and we tentatively built from RNA-seq and ESTs transcript models that are available in Supplementary file 9.

Identification of putative TFBS.

We first generated a tentative GRN for midline gene transcriptional regulation (Figure 1-figure supplement 1A) by incorporating known gene function and interactions [START_REF] Roure | An Otx/Nodal Regulatory Signature for Posterior Neural Development in Ascidians. Desplan C[END_REF][START_REF] Pasini | Formation of the Ascidian Epidermal Sensory Neurons: Insights into the Origin of the Chordate Peripheral Nervous System[END_REF][START_REF] Roure | Msxb is a core component of the genetic circuitry specifying the dorsal and ventral neurogenic midlines in the ascidian embryo[END_REF][START_REF] Waki | Genetic pathways for differentiation of the peripheral nervous system in ascidians[END_REF][START_REF] Tang | Transcriptional regulation of the peripheral nervous system in Ciona intestinalis[END_REF][START_REF] Bertrand | Neural tissue in ascidian embryos is induced by FGF9/16/20, acting via a combination of maternal GATA and Ets transcription factors[END_REF] and by formulating the following hypotheses: dorsal expression of the earliest genes, Msx and Ascl.b, may be regulated by the genes expressed earlier in DML precursors, namely Msx itself and Dlk (Notch ligand); ventral expression of the earliest genes, Msx and Ascl.b, may be regulated directly by Bmp signaling or by Bmp-activated genes/VML genes, namely Tbx2/3, Nkxtun1, Nkx2-3/5/6 and Irx.c [START_REF] Waki | Genetic pathways for differentiation of the peripheral nervous system in ascidians[END_REF][START_REF] Imai | Gene expression profiles of transcription factors and signaling molecules in the ascidian embryo: towards a comprehensive understanding of gene networks[END_REF]; and midline expression of the other genes may be regulated by all midline factors expressed before. We also considered autoregulation. For each factor or pathway (except Tox that is thought to bind DNA in a sequence-independent manner ( 75)), we assigned consensus or specificity matrices obtained for the C. intestinalis factor or orthologous gene(s) from human, mouse or fly (identified from the Aniseed database). Matrices were retrieved from Aniseed, Jaspar and CIS-BP databases [START_REF] Dardaillon | ANISEED 2019: 4D exploration of genetic data for an extended range of tunicates[END_REF][START_REF] Nitta | High-Throughput Protein Production Combined with High-Throughput SELEX Identifies an Extensive Atlas of Ciona robusta Transcription Factor DNA-Binding Specificities[END_REF][START_REF] Weirauch | Determination and Inference of Eukaryotic Transcription Factor Sequence Specificity[END_REF][START_REF] Fornes | update of the open-access database of transcription factor binding profiles[END_REF][START_REF] Yao | Schnurri transcription factors from Drosophila and vertebrates can mediate Bmp signaling through a phylogenetically conserved mechanism[END_REF] and are displayed in Figure 1-figure supplement 1B. For CRM shorter than 1 kb, sequences and their counterparts from the sister species (C. robusta and C. savignyi; P. mammillata and P. fumigata) were retrieved from Aniseed and scanned using FIMO (http://meme-suite.org/tools/fimo) with a match p-value threshold of 0.001 [START_REF] Grant | FIMO: scanning for occurrences of a given motif[END_REF]. TFBS were considered conserved when they were found close to each other (within approximately 60 bp) in the aligned sequences (alignment performed using zPicture: https://zpicture.dcode.org/ (81)). Only conserved sites were further considered in the analysis. [START_REF] Roure | An Otx/Nodal Regulatory Signature for Posterior Neural Development in Ascidians. Desplan C[END_REF][START_REF] Pasini | Formation of the Ascidian Epidermal Sensory Neurons: Insights into the Origin of the Chordate Peripheral Nervous System[END_REF][START_REF] Roure | Msxb is a core component of the genetic circuitry specifying the dorsal and ventral neurogenic midlines in the ascidian embryo[END_REF][START_REF] Waki | Genetic pathways for differentiation of the peripheral nervous system in ascidians[END_REF][START_REF] Tang | Transcriptional regulation of the peripheral nervous system in Ciona intestinalis[END_REF], more details can be found in Figure 1

  , Ascl.b, Klf1/2/4, Nkxtun3, Tox, Dlx.c and Bhlhtun1. Msx, Nkxtun3 and Dlx.c code for homeodomain-containing TFs; Ascl.b and Bhlhtun1 for bHLH TFs; Klf1/2/4

  TFBS for candidate ventral and dorsal regulators (Fig 1-supplement 1-2; Material and Methods). Ciinte.Msx-up10 contains 2 putative Msx binding sites that could account for activation in the DML. Activation by Bmp signaling in the VML could be direct (4 Bmp Responsive Elements (BRE) and 3 Smad Binding Elements (SBE)) or indirect (sites for the ventral TFs Irx.c and Tbx2/3).

  -figure supplement 1). Analysis of putative TFBS suggested activation by Msx and Ascl.b (Figure 3-figure supplement 1). Nkxtun3. A 4.8 kb region, A, immediately upstream of the gene was strongly active in VDML (Figure 3C and Figure 3-figure supplement 2). Various deletions were generated; most were active in VDML but with a lower efficiency (Figure 3-figure supplement 2).

  -figure supplement 3). Putative binding sites analysis suggested activation by the upstream factors Ascl.b and Klf1/2/4.

  figure supplement 3).

Figure 5 -

 5 figure supplement 4). While the distal region A1.2 (1.5 kb) containing one block of conservation was weakly active in VDML, the proximal one A3.1 (1205 bp) was robustly active in VDML as was a shorter version A3.2 (1034 bp) (Figure 5-figure supplement 4). The results of this section indicate strong similarities between C. intestinalis and P. mammillata for caudal PNS formation based on gene expression and gene regulation.

  -C and Figure6-figure supplement 1), the only exception being Phmamm.Nkxtun3-A3.1 that was already not strongly active in P. mammillata (Figure5-figure supplement 2). Interestingly, conserved activity was not limited to VDML: for example, activity in the anterior palp region for Ciinte.Ascl.b-do1, Ciinte.Klf1/2/4-up1 and Ciinte.Dlx.c-A2 was observed when tested in both C. intestinalis and P. mammillata (Figure2B, 3A, 3G, 6Cii, 6Ciii and 6Cvi).We next extended such functional assays in two other species, Phallusia fumigata and Ascidia mentula, that like C. intestinalis and P. mammillata belong to the ascidian Phlebobranchia order (Figure6A). A few CRMs (Ciinte.Nkxtun3-up7, Phmamm.Nkxtun3-A1 and Ciinte.Tox-int1) had weak activity, below 30%, in one or the other species. However, most tested regions were active in VDML in over 40-50% of the embryos independently of their origin (i.e. from C. intestinalis or from P. mammillata) (Figure6D-E and Figure6-figure supplement 2). Since C. intestinalis is more divergent than P. mammillata when taking the recipient species as references, this suggests that gene regulation has remained unchanged despite almost 300 My of divergence time (Figure6A).We further investigated CRM activity conservation in Phlebobranchia by isolating the genomic region upstream of Msx for both P. fumigata and A. mentula. Both regions were active in VDML when electroporated in C. intestinalis or P. mammillata embryos (Supplementary file 6).Testing the transcriptional activity of midline CRMs into four ascidian species strongly supports deep conservation of caudal midline gene expression regulation in Phlebobranchia.

  -figure supplement 2 and Figure 5-figure supplement 1). Similarly, Tox may be regulated directly by Ascl.b (Figure 3-figure supplement 3 and Figure 5-figure supplement 3.).

  Similarly to endogenous Ciinte.Msx expression, Ci-msxb-B was ectopically activated in posterior (b-line) ectoderm when the Fgf pathway was activated, and it was downregulated when the Nodal pathway was inhibited. However, upon Nodal pathway activation Ci-msxb-B was ectopically activated in posterior (b-line) ectoderm instead of anterior neurectoderm (a6.5 line); and Otx overexpression led to a repression of Ci-msxb-B instead of an activation.

  to their counterparts in C. intestinalis or P. mammillata. While Moappe.Msx and Moappe.Klf1/2/4 were expressed throughout the VDML, Moappe.Nkxtun3 expression was restricted to the posterior VDML (Figure 8B, 8D, 8E and Figure 8-figure supplement 1). Moappe.Ascl.bβ and Moappe.Tox were also expressed in the VDML but only in the posterior region and as spots (Figure 8C, 8F and Figure 8-figure supplement 1). By contrast, Moappe.Ascl.bα and Moappe.Dlx.c were not expressed in the VDML (Figure 8G and Figure 8-figure supplement 1). We also examined genes downstream of the neurogenic program, Dlk and Celf3.a (Figure 8-figure supplement 1

  and Figure 8-figure supplement 2). The other genes that we examined, Harore.Msx, Harore.Ascl.b, Harore.Klf1/2/4, Harore.Nkxtun3 and Harore.Tox, were also expressed in the VDML (Figure 8H-L and Figure 8-figure supplement 2). Among them, Harore.Ascl.b and Harore.Tox, were not expressed throughout the VDML but rather in discrete regions or spots. CESNs have been described in H. roretzi, and they express Celf3.a (also known as Etr) (Figure 8-figure supplement 2) (54-56). It is also known that their formation is regulated by the Notch pathway and the ligand Delta is expressed as spots in the VDML (57). We could also detect the expression of another Notch ligand Dlk in the VDML (Figure 8-figure supplement 2).

Figure 9 -

 9 Figure 9-figure supplement 2), both Harore.Msx-A and Harore.Tox-A were

  , H). Interestingly, the conserved active CRMs that we have identified systematically matched with open chromatin as determined by enrichment in ATAC-seq (Figure 1, 2, Figure 3-figure supplement 1-4, and Supplementary file 2) (27). Tissue-and stage-specific ATAC-seq data should certainly facilitate active CRM discovery and allow exhaustive identification of CRMs regulating a given expression pattern.

  -figure supplement 1) supports a model of maintenance of gene expression by autoregulation whereby Msx protein produced through the early proximal CRM activates late Msx expression via the distal CRM (Figure 1-figure supplement 2). Second, we could classify the genes in two classes: Msx and Ascl.b with uncoupling of regulation in dorsal and ventral midlines (Figure 1 and 2); and Klf1/2/4, Nkxtun3, Tox and Dlx.c regulated in a 'pan-midline' manner (Figure 3-figure supplement 1-4). These observations are in agreement with previous predictions based on regulatory interactions (42,44-46): Msx and Ascl.b are upstream regulators that integrate inputs from the dorsal and ventral midline inducers and regulate the expression of the other four downstream factors. Surprisingly, we did not identify VML specific CRMs. While the reason is still unclear, a possible explanation would be that there is a physical overlap between VML and DML CRMs with the DML being embedded in the VML CRM. Prediction of TFBS for putative trans acting factors in the Msx VDML minimal CRM (Ciinte.Msx-up10) supports this hypothesis: binding sites for dorsal and ventral factors are intermingled or overlapping (Figure 1-figure supplement 2). Targeted mutagenesis of these putative binding sites should enable testing such an hypothesis. Third, analysis of conserved TFBS in small active CRMs provides interesting hypotheses that will need to be tested experimentally using finer and possibly quantitative approaches. While both Msx and Ascl.b are likely to integrate initial inducing cues, they do not seem to be regulated similarly: Msx is possibly a direct Bmp signaling target in the VML like vertebrate Msx in some tissues (58,59), but Ascl.b is not (Figure 1-figure supplement 2 and Figure 2-figure supplement 1). Klf1/2/4 and Nkxtun3 are possibly directly activated by Msx, and Tox by Ascl.b. Finally, it is most likely that additional TFs remain to be identified to understand gene regulation in the VDML since the activity of several CRMs and deletions cannot be explained using the TFs that we currently know.

  figure supplement 3). These observations are in agreement with conserved

  and 10). First, in both Molgula and Halocynthia, the genes Ascl.b and Tox were not expressed throughout the VDML but had a spotted expression. Such an expression pattern is correlated with the number and location of future PNS neurons. As shown in Figure 8-figure supplement 1-2 and already known for Halocynthia, presumptive neurons labeled by Celf3.a expression are present in a very small number and only in the posterior tail, at least on the ventral side, when compared to Ciona (42,55-57). This would suggest a variation at the level of the Stolidobranchia family and that the function of Ascl.b and Tox would possibly be different from the other 'pan-midline' transcription factors and from their orthologs in Phlebrobranchia. Interestingly, in Phlebobranchia, we have seen above a possible direct regulation of Tox by Ascl.b, suggesting that PNS formation in ascidians could be controlled by the following VDML sub-networks: Ascl.b and Tox on one side, and Msx, Klf1/2/4, Nkxtun3 and Dlx.c on the other side. Second, in Molgula, Nkxtun3 expression was restricted to the posterior VDML. Third, Moappe.Dlx.c was not expressed in the VDML while palp expression was conserved (Figure 8-figure supplement 3). These two latter observations suggest a genus-or species-specific variation.

Figure 1 .

 1 Figure 1. CRMs controlling Ciinte.Msx expression in VDML. (A) Snapshot of the

Figure 2 .

 2 Figure 2. CRMs controlling Ciinte.Ascl.b expression in VDML. (A) Snapshot of

Figure 3 .

 3 Figure 3. CRMs with activity in the tail epidermis midlines for Ciinte.Klf1/2/4,

Figure 4 .

 4 Figure 4. Expression and regulation of caudal PNS genes in Phallusia

  -figure supplement 1A). (B-L) Expression of the P. mammillata orthologs of the C. intestinalis caudal PNS genes. In situ hybridization of a late neurula for Msx (B), at early tailbud stages for Ascl.b (C), Klf1/2/4 (C), Nkxtun3 (D), Tox (F), Bhlhtun1 (G), Dlx.c (H) and Dlk (I); and at mid/late tailbud stages for Atonal (J), Pou4F (K) and Celf3.a (L). (M-Q) Expression of Phmamm-Klf1/2/4 at early tailbud stages in control embryos (M), following electroporation of pFog>Ciinte.Admp (N) or pFog>Ciinte.Noggin (P); or following treatment with Bmp4 protein (O) or Dorsomorphin (Q). (R-U) Expression of Phmamm.Pou4F at late mid/late tailbud stages in control embryos (R), following treatment with Bmp4 protein (S), DAPT (T) or a combination of Bmp4 and DAPT (U). Embryos are shown in lateral view with dorsal to the top and anterior to the left. Scale bars: 50 μm.

Figure 5 .

 5 Figure 5. CRMs with activity in the tail epidermis midlines for Phmamm.Msx,

Figure 6 .

 6 Figure 6. Conserved VDML activity during enhancer swaps in Phlebobranchia.

Figure 7 .

 7 Figure 7. Minimal CRMs are not robust to phylogenetic and genetic challenges.

Figure 8 .

 8 Figure 8. Overall conservation of midline gene expression in Stolidobranchia

Figure 9 .

 9 Figure 9. Divergence in gene regulation in Stolidobranchia ascidians. (A-F)

Figure 10 .

 10 Figure 10. Summary of comparative results obtained in the present study. For

Figure Supplements.

  Figure Supplements.

Figure 1 -

 1 Figure 1-figure supplement 1. A) Provisional GRN for caudal PNS specification in

Figure 1 -

 1 Figure 1-figure supplement 2. A) Identification of putative TFBS for candidate

Figure 2 -figure supplement 1 .

 21 Figure 2-figure supplement 1. Identification of putative TFBS for candidate

Figure 3 -

 3 Figure 3-figure supplement 1. CRMs controlling Ciinte.Klf1/2/4 expression in

Figure 3 -

 3 Figure 3-figure supplement 3. CRMs controlling Ciinte.Tox expression in

Figure 3 -

 3 Figure 3-figure supplement 4. CRMs controlling Ciinte.Dlx.c expression in

Figure 4 -

 4 Figure 4-figure supplement 1. Expression pattern of caudal PNS genes in

Figure 5 -

 5 Figure 5-figure supplement 1. CRMs controlling Phmamm.Msx expression in

Figure 5 -

 5 Figure 5-figure supplement 3. CRMs controlling Phmamm.Tox expression in

Figure 5 -figure supplement 4 .

 54 Figure 5-figure supplement 4. CRMs controlling Phmamm.Dlx.c expression in

Figure 6 -

 6 Figure 6-figure supplement 1. Conserved VDML activity during enhancer

Figure 6 -

 6 Figure 6-figure supplement 2. C. intestinalis and P. mammillata midline CRMs

Figure 8 -figure supplement 1 .Figure 9 -figure supplement 2 .

 8192 Figure 8-figure supplement 1. Expression pattern of caudal PNS genes in

1 KhL9

 1 ||| ||:|:|:||| ||:|||||::|:| :||::|:||:|||||| Cisavi TTATGAATCGACCATCGC--GCGTTTGATGCGC-----------TGACAATGGCGTCGTA Ciinte ACTTAACAATAGTCTGGGGCCTATGATGTCATAATGCCCATTTGGTGCACTCGCGTGCTG |:||||||||:||||||||:||:||||||||||||:||: |||:|||||||||| Cisavi AGTTAACAATCGTCTGGGGTCTGTGATGTCATAATTCCA-------GCAGTCGCGTGCTG Ciinte TCGCCCAAAATCGCAATTAACTCGATGACACTGGCAGC-ATATGACGTCATAAAATGGTT ||||||:|||:||:|||||||||:::|:::|::|:::| |||||||||||||||::::| Cisavi TCGCCCGAAAACGTAATTAACTCAGAGCTCCAAGGGTCTATATGACGTCATAAACAAAT-Ciinte GCCAGGGTCCCGGACCGAGACAGTAATTCACTTTTATGACCCGAAT ||||:| :|:||||::|||||||::||||:|||||||||::|:|| Cisavi -CCAGCG-ACTGGACACAGACAGTCCTTCATTTTTATGACTAGCAT Bmp Responsive Element (GRCGNC) .25.v1.A.SL1-1 KH.L9.13.v1.B.ND1-1 Ascl.b KH.L9.13.v2.B.SL2-1 KH.L9.21.v1.R.SL1-1 KH.L9.21.v2.B.SL1-: ||:||||:|||| |:||:|||::||| :|:|:||:|:|::|||||||||||||| Cisavi C-CGGGATCCATTC-----TTACGTCAAAATGCGCTTTACGACGGAGCAGGTGGAATTCG Ciinte AGAACTTGGAAACAAACACCGCGATCACCGCGATTATGACGTCAAATGTTTAAACAATCG :|||||||:|:|||||||| ||||||||||| |::|||||||||||:|| Cisavi GGAACTTGAAGACAAACAC------------GATTATGACGT-AGCTGTTTAAACAAGCG Ciinte ATGTCTGCATATTTCGACGCGAATGAATATCTTAGAAATCATCGTAGATTATTTTATAAT ||| ||||||| |||:|||||||:|::|:|:|||:::::|::|| Cisavi ATG----CATATTT--------------ATCATAGAAATAACAGAAAATTGGCGCACTAT Ciinte GTCTATTATG----ACGTAACATTCGATTAAT-------AATCGCGCCTGTTTGAAAGAT ::|:||:||: |||::|::|::||:|||: ||||::::||:|:|:||:::| Cisavi CACCATAATATTCAACGCGAAGTCAGACTAAGCACTTTAAATCAGAACTTTCTTAATATT Ciinte T------CTTTTGTTTTTAATAAAAATTAAAACTTTTATGACCAA-ACAGATTGTTTTAT | :|:|:|:|||||||||:|||::|:| ||||||||:||| |||||||||||||| Cisavi TGATTAATTCTGGCTTTTAATAATAATCGAGA-TTTTATGATCAAGACAGATTGTTTTAT Ciinte GACGTCATAATCAGTCAACAATGACAACTTTAATCCGTGTTTATTACTATGATGTAACAA ||::::|||||:|::||:|:|||:|||:||||||||:||||||||:||:||||::||||| Cisavi GATTATATAATGACACAGCGATGTCAAATTTAATCCTTGTTTATTGCTGTGATTAAACAA Ciinte TGGTAGATTATGTAAACAA--TAGAATCACACACCGTATTGTTACGTCACAAATTCT ||:::::||:||||||||| ::||:||::|| |:|:||||||||||||:||||||: Cisavi TGAGCTTTTGTGTAAACAAGGCCGAGTCCTAC-CTGCATTGTTACGTCATAAATTCC Putative ventral regulators: Smad Binding Element (AGAC)

  ||::||||||||:||::::|||||||||||||::||||:|| |||||||||||| Cisavi CACTTCTGCCTGATTTATTGCCTTGCTGCTTTGATGTGTCTCGACTTCGGAGATTTTTCG Ciinte ACTTGCGGCAGATGTTCCATTCTCTAAACAATGGCGGA---------GATGAATTATGCC |||||||||||||||||::||||||||||||||||||| |||||||:||::| Cisavi ACTTGCGGCAGATGTTCTCTTCTCTAAACAATGGCGGAAACGCTGGAGATGAATGATCAC Ciinte TGATATTTTCGCCACGCCGTAGGTTTCTTTGCTTTCCCAAGGCGCGAAATTGGTTTTCTC ||:||||| |:|::|||::|:||||||||||||||:|:::|::|||:|| :|:|||:|| Cisavi TGCTATTT-CTCAGCGCAATGGGTTTCTTTGCTTTGCAGTGTTGCGGAA--AGCTTTATC Ciinte TCTTTTCTACGCTTTGTGTTTTCTATCCCACGTCGAGATTTTTGTCATCGCTTCGTGTTT ||||::::::|::||||||::|:| :::||:|||||||||||||||||||:::|:||||| Cisavi TCTTCGTGGTGTATTGTGTGCTTT-CGTCAGGTCGAGATTTTTGTCATCGGAGCTTGTTT Ciinte GCTTTTCCGTAATGTGTTTGTCTTGCTAATTCTCGAACAGAAGGCGAAATGTAAATACAC |:|||:||||||||||||||:|||||||||||:|||||||||||||:::||||||||::| Cisavi GTTTTACCGTAATGTGTTTGCCTTGCTAATTCACGAACAGAAGGCGGTGTGTAAATAATC Ciinte GTTGCCGA--CGCGACACAAACTGCCGTTGTTCATCAACACGTATTGACAGC--GACAAA :::||||: :|:||:|:|||||::|::|::||:::||||||::|::||:|| ||:::| Cisavi TCCGCCGGGTTGTGATATAAACTAGCACTTCTCGCAAACACGATTCCACGGCCGGATCCA Ciinte CGCTGGTCCAACCAGCCAATTCTGCACCGTTTCCGTCGCCCGAAGTCTCGAGGAGAGAAT ||:|| ||:|||:|||:||||||||:||:|::|:::|:|||:|:|:|||| Cisavi CGTTG----------

  :||::|||||||||||||:||||||||||||||||||: Cisavi TCGGTGATTTGTCGCCAATTGATTTGTTTGTTTCTCTGTTTATCTGTCGCGATCT-----Ciinte GCTCGATGCCGCATCAAACCACCTTTAACTCGCCATTAGCGCAACGCG-GGTGACGCTTC ::||:|::||:||:|:||||:|||:::|||||||:|:|:::|: |||:||||:|: Cisavi -----GGGCGGTTTCGAATCTCCTTGAACCTCCCATTAGTGGATGACATGGTAACGCATT Ciinte ---------ACT-----------AATACCTCCGCACCACCAAGCACGAGGTGT-------||| |::||:::::||||::||::::||||::|: Cisavi GGATCTGTGACTGTGAAAGATGGAGCACTGTAACACCCTCATAAGCGAGACGCTCTGGGA Ciinte ---ATGAATGTATACTGGAATAT---TAAGATCGGCGAAATGGGTTGTTTTCAGG---AC :||:||:::||::::::||: |||:|::||| |:||||::|:|:|::|::||||||:||::||:|| |:||:|::|:|:||:||:|:|:::|: Cisavi ACTCGACATGTTTGTTAGCCTTG-GTTTCGATTTGAGTGAAGGTTATTTTTATTTGTTAT Ciinte ---------AAAAATGCGTCATTCCGCGTCCGTAATTA----ACCACGGCGATGGTTTTT |||||||::|::||::: ||||| |||::|:::|||:||:| Cisavi AATTAAACTAAAAATGTATATTTTAA-------AATTATAGGACCGTGAAAATGCTTGT-Ciinte CGATTAAGATTGGAAAT-----------------------CGAAGCGACTTTACG---CC :|||| |:|:||||| |||:

  -TGTTTACGGTGCTCTG-CTAACGTGCTGTGATTGTTTA ||:|||::|:|:||||||| :|||||||||::|::: |:|||||||:||||::|||:| Phfumi ATATCTACAACAGCCAACAGCACGTTTACGGTTTTTCATCCAACGTGCCGTGACCGTTCA Phmamm TGGCGCTTAATTGTGTGAAGAAAATACTAGTGTCCATAAAATACAAAACAACTACCCAAT :|||:|||:|||||:||||||||||::|:|:|||::||| |::||:||||||||:|||| Phfumi CGGCACTTTATTGTTTGAAGAAAATTGTGGCGTCTCTAA--TTTAAGACAACTACGCAAT Phmamm CACTTTTAAG-GCAATATAATTTTCGCATGTGCGTTTGATCAGCTCGTTAATTTCAAATC |||:|||||| ||||||:|||:|||||:|||:|||||||||:|||||||||:|||:|||| Phfumi CACCTTTAAGTGCAATAAAATCTTCGCTTGTACGTTTGATCGGCTCGTTAAGTTCGAATC Phmamm GCGGCGTCGACAAGAACATAAAACTCGTCTGCTCGACGTCTGACGCGTGTCTGGCACCAC |||||||||||:||||:| ||||||:|||||||:|||||||||||||||||||||||:|| Phfumi GCGGCGTCGACGAGAATA-AAAACTTGTCTGCTGGACGTCTGACGCGTGTCTGGCACAAC Phmamm CCTGCTTACAGAAGTCTACGTCTTGTTTTCTTTATTCCATAAATACCTGGCCCCCAAAAA ||||:||||::||||||:::||||||||:|||||:|:: |:::||:|||:::::|:||:| Phfumi CCTGATTACCAAAGTCTGTCTCTTGTTTGCTTTACTTT-TCGCTAACTGAAAAACCAACA Phmamm ATGTTGAAACAACTTTTTTGTGA-----ATATAAAATAGAAATAACCAAAC ||:||:|||:||::::|||||:: |:||:|||:|:|:|||:|||||| Phfumi ATATTCAAAGAATACATTTGTCGCAATTAGATTAAAAAAACATACCCAAAC Bmp Responsive Element (GRCGNC)

  |::|:|:||||||||||::|:::| ||:||||||||||||:|||:: Phfumi ATGAACACAT---ACGCCAGGAGGAAATACTGTGATT--GAGATTGTTTATTTACCTTTT Phmamm TAAATTCGCTCAGGTCGTGTTTGTTT---GTAGTGCACGGGGCACTGCCAGCCAGGGTGG ::||||||:||:||:||||||||||| |:||::||||||||::||||:|||||:|||| Phfumi CGAATTCGGTCGGGGCGTGTTTGTTTTTGGCAGCACACGGGGCTTTGCCCGCCAGTGTGG Phmamm CGCCCATATAATGACAGGTTTACCGTCCTTGCACCTGCCGGTCGAGGGGCAGGTGCTCCG ||||:|||||||||||:||||::|:||||||||||||||||||||||||||||||:|||| Phfumi CGCCAATATAATGACATGTTTTTCTTCCTTGCACCTGCCGGTCGAGGGGCAGGTGTTCCG Phmamm CCACGCACCAATGAACACTAAATTATTTATTATATTTG-TATTGATTCCGAATTTCCAAT :|:||||:||:||||||||:|||||||||||||||||: |:|||||||:||||||:::|| Phfumi ACGCGCATCACTGAACACTGAATTATTTATTATATTTTCTGTTGATTCTGAATTTAGTAT Phmamm CCAACTCAGATTTGGTCGGAACTGTTAACAA---TGTTACACAAATTAAAAGGCTTTTGA |::| |:|||:|::::|:|:|:||||| |:||::||||||:||::|:::|||:| Phfumi CAGA-----ACTTGTTAAACAATATCAACAAAATTTTTCAACAAATGAACTGAAATTTAA Phmamm CTA-------TTGTTTTGTTTTATTTT----AAAATCC--------------------------CCTCCGCCGCATTTGAACAAAAAGAAAAAGACAATGTAATA---------AGA :::::|::|::||::||:||:|||:||||:: ||||:||: --------------AATATTTGT ||||:::||:|||| |:|||:::::||||:::|:||| ||:||::|| Phfumi AAATGTCAAGACAGGTTTGCATTTAAAAATTACAAAATTAATAGCTAGAACAAAATAAGT

  .CG.MTP2014.S236.g05894.01.t Phmamm.CG.MTP2014.S236.g05895.01.t Msx Phmamm.CG.MTP2014.S236.g05895.02.t Phmamm.CG.MTP2014.S236.g05896.01.t

	S236	167600	168000	168400	168800	169200	169600	170000	170400	170800	171200	171600	172000	172400	172800	173200	173600	174000	174400	174800	175200	175600	176000	176400
	20
	ATAC Mid Neurula B
	0
	Phmamm cDNAs							Msx
	Phmamm CRMs
					UP
				LED+UR
			LED	Pm-msxb-b6.5 line
	transcripts
	100
	Phallusia mammillata/
	Phallusia fumigata
	conservation score
	30
															% of embryos stained in VDML
									UP		(n=708, N=6)
								LED+UR
													0 %	20 %	40 %	60 %		80 %		100 %

Phmamm

Tox

  Phmamm.CG.MTP2014.S258.g06244.01.t Phmamm.CG.MTP2014.S258.g06244.02.t Phmamm.CG.MTP2014.S258.g06245.01.Phmamm.CG.MTP2014.S258.g06246.01.t Phmamm.CG.MTP2014.S765.g11802.01.t Phmamm.CG.MTP2014.S765.g11803.01.t Tox Phmamm.CG.MTP2014.S765.g11803.02.t

	S258	12000	13000 58000	14000	15000 59000	16000 60000	17000 61000	18000 62000	19000 63000	20000 64000	21000 65000	22000 66000	23000 67000	24000 68000	25000 69000 26000	27000 70000	28000 71000	29000 72000	30000 73000	31000 74000	32000 75000	33000 76000	34000 77000	35000 78000	36000 79000
	S765
		20
	ATAC Mid Neurula B
	ATAC Mid Neurula B
		0
	Phmamm CRMs Phmamm CRMs										A	A
									A3		A1			A1
									A3.1	A1.1	A2
	Phmamm cDNAs			Klf1/2/4							A2
	transcripts
	Phmamm cDNAs
	transcripts
	100
	Phallusia mammillata/
	100
	Phallusia fumigata
	Phallusia mammillata/
	conservation score
	Phallusia fumigata
	conservation score
		30
																	% of embryos stained in VDML
	A		A3
										A			(n=870, N=3)
									A1			(n=210, N=1)
																	% of embryos stained in VDML
									A2			(n=142, N=1)
									A3			(n=279, N=3)
									A3.1			(n=33, N=1)
															0 %	20 %		40 %		60 %	80 %	100 %
																		Phmamm.Klf1/2/4
																0 %	20 %	40 %		60 %		80 %	100 %
	Phmamm CRMs
	Phmamm cDNAs
	transcripts
	100
	Phallusia mammillata/
	Phallusia fumigata
	conservation score
	A		A1
																	% of embryos stained in VDML
										A			(n=627, N=3)
										A1			(n=186, N=2)
										A2			(n=108, N=1)
	A3									A3			(n=185, N=2)
									A3.1			(n=81, N=1)
									A3.3			(n=142, N=1)
															0 %	20 %		40 %		60 %	80 %	100 %
																	Phmamm.Nkxtun3

3 Nkxtun3 Phmamm.CG.MTP2014.S486.g09311.03.t Phmamm.CG.MTP2014.S486.g09312.01.t Nkxtun3 Phmamm.CG.MTP2014.S486.g09312.02.t Phmamm.CG.MTP2014.S486.g09313.01.t Nkxtun3 Phmamm.CG.MTP2014.S486.g09314.01.t S486 ATAC Mid Neurula B t Klf1/2/4

Acknowledgments.

We thank EMBRC-France (Banyuls-sur-mer and Roscoff marine stations) and G. Diaz (Port-Vendres) for providing animals. We are grateful to C. Labrune, J.M. Amouroux and F. Monniot for help in identifying M. appendiculata. Many thanks to the Phallusia genome consortium (H. Yasuo, A. McDougall and P. Lemaire) for access to various resources (genomes, transcriptomes and cDNA clones).

Supplementary file 5). The topology of the Msx locus is strikingly similar between C. intestinalis and P. mammillata: there is local synteny (the gene upstream of Msx is orthologous: KH2012:KH.C2.808 in C. intestinalis and Phmamm.g00005894 in P. mammillata) and the small intergenic region contains a proximal CRM active early in DML precursors and a distal CRM active late in VDML (Figure 1 and Figure 5-figure supplement 1). TFBS analysis of the LED fragment suggested that Phmamm.Msx could be regulated like Ciinte.Msx: a direct target of Bmp signaling in the VML and of Msx in the DML (Figure 5-figure supplement 1).

Klf1/2/4.

We first isolated a 6.7 kb conserved region immediately upstream of Klf1/2/4, A, which was strongly active in VDML (Figure 5B, Figure 5-figure supplement 2 and Supplementary file 5). When subdivided into 3 large pieces, 2 were active in VDML, suggesting a possible redundant regulation (Figure 5-figure supplement 2). The proximal region A3 (2.5 kb) was the region with strongest activity.

Nkxtun3.

We first isolated a 7.2 kb conserved region 2 kb upstream of Nkxtun3, A, which was strongly active in VDML (Figure 5C, Figure 5-figure supplement 2 and Supplementary file 5). Similarly to Phmamm-Klf1/2/4, 2 out of 3 sub-regions of A were active in VDML (Figure 5-figure supplement 2). In this case, a spatial difference of activity was observed: the distal region A1 (2.5 kb) was mainly active in the posterior half of VDML while the proximal region A3 (2.8 kb) was active throughout VDML. This latter region could be reduced to 1.5 kb (A3.1) with maintaining robust activity. However, a shorter fragment A3.3 (0.6 kb) was barely active.

Tox.

sites for Msx makes it a likely activator. Up11 is a smaller derivative of up7 (235 bp fragment long) that behaves as a minimal VDML enhancer (Figure 3B) and is deleted in the up8 region (inactive). It is located 2.4 kb upstream of Klf1/2/4, corresponds to an ATAC-seq enrichment detected from late gastrula stages (Supplementary file 2) and contains the 2 Klf1/2/4 sites, suggesting it might be involved in autoregulation/maintenance. Note that the size of the highlighted site for of a given TF may vary depending on the matrix used. intestinalis, P. mammillata, M. appendiculata and H. roretzi. For each gene, the main sites of expression from late neurula stages in C. intestinalis [START_REF] Roure | Msxb is a core component of the genetic circuitry specifying the dorsal and ventral neurogenic midlines in the ascidian embryo[END_REF][START_REF] Imai | Gene expression profiles of transcription factors and signaling molecules in the ascidian embryo: towards a comprehensive understanding of gene networks[END_REF] are schematized on the left. For the other species, identical pattern is represented as a green circle, expression in part of the tissue as an orange circle, and absence of expression as a red circle. For simplicity, developmental timing has not been described, but it has been taken into account for comparisons. For example, Ciinte.Ascl.b is expressed in the palps forming region from initial tailbud to late tailbud stages, while Moappe.Ascl.bα was detected in the palps only at late tailbud stages. This difference was depicted by an orange circle.

Supplementary Files.

Supplementary file 1. List of all genomic regions tested in the present study.

Supplementary file 2. Genome browser view for each locus of the 7 caudal PNS neurogenic TFs in Ciona robusta. Tested CRMs were added to the data extracted from the Aniseed website (https://www.aniseed.cnrs.fr/) [START_REF] Dardaillon | ANISEED 2019: 4D exploration of genetic data for an extended range of tunicates[END_REF]. Note that while VDML activity is rather robust for Phmamm.Bhlhtun1-A, it was not considered further since this activity was restricted to the very posterior cells of the midlines at the tail tip.

Supplementary file 3. Activity of various genomic regions for the gene

Supplementary file 5. Genome browser view for each locus of the 7 caudal PNS neurogenic TFs in