Regulation of anterior neurectoderm specification and differentiation by BMP signaling in ascidians

Three palps make the most anterior structure of the ascidian larva. These ectodermal derivatives have both a sensory and adhesive functions essential for metamorphosis. They derive from the anterior neural border and their formation is regulated by signaling pathways such as FGF and Wnt. Since they also share gene expression profiles with vertebrate anterior neural tissue and cranial placodes, their study should shed light on the emergence of the unique vertebrate telencephalon. Here, we show that BMP signaling regulates two phases of palps formation in Ciona intestinalis. During gastrula stages, the anterior neural border marked by Foxc is specified in a domain of inactive BMP signaling, and activating BMP prevented its formation. Later on, inhibiting BMP led to the formation of a single large palp, most likely of dorsal identity. Our results indicate that BMP signaling regulates papilla vs inter-papilla fate decision within the palps forming region. Finally, we showed that modulating BMP signaling led to similar palps phenotypes in another ascidian species Phallusia mammillata. This led us to screen transcriptomic data and identify novel palps markers. Collectively, we provide a better molecular description of palps formation in ascidians that will be instrumental for comparative studies within ascidians and between ascidians and other chordates.


Introduction 34
Ascidians (or sea squirts) belong to a group of marine invertebrates, the tunicates, that is the 35 sister group of vertebrates. This phylogenetic position associated with a stereotyped 36 embryonic development with few cells puts ascidians as interesting models for 37 developmental biology and comparative approaches to address questions regarding 38 chordates evolution and the emergence of vertebrates. Ascidians have a biphasic life cycle: 39 following external development, the embryo gives rise to a swimming tadpole-like larva with 40 typical chordate features (notochord, dorsal neural tube) that is going to attach to a 41 substrate before metamorphosing into a sessile adult ascidian with a radically different body 42 plan, a 'bag' with two siphons. Metamorphosis is controlled by a specific organ, the palps 43 (also referred to as the adhesive organ or the adhesive papillae), that is located at the 44 anterior end of the larva. The palps are a specialized part of the ectoderm that has adhesive 45 and sensory properties. They enable the larva to select a suitable substrate for 46 metamorphosis, hence a chemo-and/or mechano-sensory function, and to attach to it 47 through the secretion of adhesive materials. It contains at least four cells types whose 48 specification and function have not yet been deciphered in details (Johnson et al., 2020;49 Zeng et al., 2019). Three cell types -the ciliated sensory neurons, the collocytes (containing 50 vesicles filled with adhesive material), and the axial columnar cells (ACCs) (myoepithelial 51 cells controlling palps retraction following adhesion) -are elongated cells forming a 52 protrusion. Three protrusions or papillae (two dorsal papillae that are bilaterally 53 symmetrical, and a ventral papilla located at the midline; Fig 1C) make up the palps and are 54 separated by the fourth cell type, the non-elongated inter-papillae cells. 55 Palps belong to the peripheral nervous system and have been instrumental for proposing 56 evolutionary scenarios on the nervous system in chordates. In the ascidian Ciona intestinalis, 57 palps cell lineage and topology, together with gene expression data and functional studies, 58 have shown affinities with anterior derivatives of the vertebrate nervous system, the 59 olfactory placodes and the telencephalon (Cao et al., 2019;Horie et al., 2018;Hudson et al., 60 epidermis midline with a similar temporal dynamic (Fig 2G,2I,2K and 2L), and in a group of 143 cells of the anterior sensory vesicle at mid-tailbud stages (Fig 2K and 2L). We validated the 144 specificity of these results by modulating BMP pathway: when embryos were treated with 145 BMP2 protein, P-Smad1/5/8 was ectopically detected in the entire epidermis at early 146 neurula stages, while no staining was observed following inhibition using the 147 pharmacological inhibitor DMH1 (Fig 2D and 2E). To precisely relate the location of active 148 signaling in the ectoderm and palps precursors, we performed double staining: P-Smad1/5/8 149 and in situ hybridization for the early palp markers Foxc and Foxg (Fig 2G-K). At late gastrula 150 stages, P-Smad1/5/8 abutted Foxc expression domain, confirming that palps precursors 151 were specified in a BMP-negative domain (Fig 2G and 2H). Later, we observed P-Smad1/5/8 152 in the median Foxc expression domain at mid neurula stages (not shown) and in the median 153 part of the U-shaped Foxg expression domain (Fig 2I and 2J), corresponding presumably to 154 the future ventral palp. This was confirmed by co-expression of P-Smad1/5/8 and Foxg in 155 ventral cells in late neurulae and early tailbuds. (Fig 2J and 2K). 156

BMP inhibition participates in ANB definition 158
We next tested whether BMP inhibition was sufficient to induce an ANB fate. When BMP 159 signaling was blocked either by overexpression of the secreted inhibitor Noggin or by 160 treatment with the BMP receptor inhibitor DMH1, Foxc expression at late gastrula stages 161 was unchanged ( Fig S1). The fact that Foxc was not ectopically expressed following BMP 162 inhibition could be explained by an incomplete BMP blockade. However, DMH1 treatment 163 led to undetectable P-Smad1/5/8 levels ( Fig 2E). Alternatively, it could be that the number of 164 cells that are competent to become ANB in response to BMP inhibition could be restricted to 165 the cells already expressing Foxc. Foxc expression and palps fate are regulated by FGF 166 signaling following neural induction and cell fate segregation (Wagner and Levine, 2012). We 167 thus aimed at increasing the number of cells competent to form ANB by early activation of 168 FGF signaling using treatment with recombinant bFGF protein, and testing the effects of 169 BMP pathway modulations in this context. As expected, bFGF treatment from the 8-cell 170 stage neuralized the entire ectoderm as revealed by the ectopic expression of the neural 171 markers Otx and Celf3/4/5/6 and the downregulation of the epidermal marker Tfap2-r.b at 172 late gastrula stages (Fig 3 and S1). Foxc behaved somewhat unexpectedly: it was either 173 ectopically expressed or repressed (Fig 3D). The repression of Foxc might be explained by the 174 fact that FGF/Erk is downregulated in the palps lineage during gastrulation (Wagner and 175 Levine, 2012), hence our continuous treatment might inhibit Foxc expression. Nevertheless, 176 when BMP pathway was inhibited in addition to FGF activation, Foxc was strongly expressed 177 as a cup covering the anterior end of the embryo, including the ventral epidermis ( Fig 3F). 178 Importantly, the loss of Foxc following BMP activation using recombinant BMP2 protein 179 treatment was specific to this gene and did not result from neural tissue inhibition as in 180 vertebrates since the neural markers Otx and Celf3/4/5/6 were still expressed in the CNS but 181 downregulated in the ANB (Fig 3 and S1). Reciprocally, BMP inhibition was not sufficient to 182 lead to ectopic neural tissue formation. This is in agreement with similar data produced in 183 the distantly related ascidian Halocynthia roretzi (Darras and Nishida, 2001). These 184 observations show that BMP limits the domain of Foxc expression and ANB formation. 185

186
The BMP signaling pathway regulates palps formation following ANB formation 187 We next examined whether modulating BMP had any impact on palps formation besides 188 ANB specification. We thus performed whole embryo treatments starting at progressively 189 later stages of embryonic development and examined the early marker Foxc and the late 190 marker Isl (Fig 4). Activating BMP at early gastrula stages (St. 10) partially repressed Foxc,191 and abolished ventral palp formation. Mid-gastrula (St. 12) treatment had no effect on Foxc 192 and repressed ventral palp at a lower frequency than the earlier treatment. Later treatments 193 did not change Isl expression. Inhibiting BMP had no effect Foxc expression as for the earliest 194 treatment (Fig 3 and S1). Isl, that is normally expressed as 3 spots, had a U-shaped 195 expression. This phenotype was much less frequent when the DMH1 treatment started at 196 late neurula stages (St. 16). 197 We next focused on understanding the U-shape pattern. It reminded us endogenous Foxg 198 expression (Liu and Satou, 2019): at neurula stages, Foxg was expressed in the future palps 199 following a U-shape that gradually converted into a 3-spots pattern at tailbud stages that 200 prefigures the three papillae protrusions (Fig 5A-D). It thus seems that inhibiting BMP 201 prevents the refinement of Foxg expression. Accordingly, Foxg expression was U-shaped 202 following DMH1 treatment ( Fig 5E). Interestingly, knockdown of Sp6/7/8/9 leads to a U-203 shaped Foxg expression (Liu and Satou, 2019). Since Sp6/7/8/9 and Foxg are initially partially 204 co-expressed before showing exclusive patterns, it has been proposed that Foxg restriction 205 to the future protrusions is the result of repression by Sp6/7/8/9. We thus determined 206 In DMH1 embryos, Pou4 was expressed all around the Isl cells like in the dorsal palps. This 238 suggested that the Cyrano protrusion may have a dorsal identity. In support of this 239 interpretation, we found that the expression of the homeobox transcription factor Msx, that 240 we found transiently expressed in the future ventral palp at the onset of Isl expression (Fig  241   6L and 6M), was lost following BMP inhibition (Fig 6N and 6O). 242 243 Palps formation is similarly regulated by BMP in P. mammillata 244 We aimed at determining the conservation of the role of BMP in palps differentiation by 245 examining embryos of the ascidian P. mammillata that belongs to the same family as Ciona, 246 the Phlebobranchia, but with a significant divergence time (275 My) ( Fig 7A) (Delsuc et al., 247 2018). First, we determined that BMP signaling was active is the ventral part of the embryo 248 with a similar dynamic to Ciona as revealed by P-Smad1/5/8 immunostaining ( Fig S2). Next,249 we identified single orthologs for Celf3/4/5/6, Pou4 and Isl genes, that were all expressed in 250 the palps (Fig 7) (Chowdhury et al., 2022;Coulcher et al., 2020;Dardaillon et al., 2020). 251 Treatment with recombinant BMP2 protein from the 8-cell stage abolished expression of all 252 three markers in the palps, like in Ciona (Fig 7). Following DMH1 treatment from the 8-cell 253 stage, both Celf3/4/5/6 and Isl were expressed in the palps territory following a U-shape 254 pattern like in Ciona but not in all cases. For a large fraction of embryos, the pattern 255 appeared as two bars of intense staining resembling the U-shape but without the ventral 256 part. This phenotype that we did not observe in Ciona might reveal some differences in the 257 role of BMP in the two species. 258 Given the overall similar effects on palps formation after alterations of BMP signaling, we 259 sought to identify novel palps molecular markers by using a dataset previously generated in 260 P. mammillata (Chowdhury et al., 2022). We had generated, at several developmental 261 stages, RNA-seq data for whole embryos treated with recombinant BMP4 protein and/or 262 DAPT, a pharmacological Notch inhibitor. We identified 1098 genes repressed by BMP 263 signaling at least at one developmental stage (Table S1). In this list, we found the orthologs 264 for 11 well defined Ciona palps markers; and 4 of them (Otx, Isl, Atoh1/7 and Celf3/4/5/6) 265 were described as expressed in the palps lineage in Phallusia (Coulcher et al., 2020;266 Dardaillon et al., 2020). Using Gene Ontology analysis, we selected a list of 53 genes 267 encoding developmental regulators (transcription factors and signaling molecules) or 268 involved in neural tissue formation, and examined their expression patterns (Table S2). By 269 searching the Aniseed database (Dardaillon et al., 2020) and our previously generated 270 expression data (Chowdhury et al., 2022;Coulcher et al., 2020), we identified 12/26 genes 271 expressed in the palps. By performing in situ hybridization for 27 extra genes, we discovered 272 7 novel palps markers whose expression is shown in Fig 8. 273 Surprisingly, by examining the expression data generated previously, we found that some 274 genes with palps expression were up-regulated by BMP in our dataset, such as Chrdl and Nos 275 (Table S2 and Fig 8). To have a broader view of the potential effect of BMP signaling on gene 276 regulation in the palps, we gathered, from previous publications (Chen et al., 2011;277 Chowdhury et al., 2022;Coulcher et al., 2020;Joyce Tang et al., 2013;Kusakabe et al., 2012;278 Liu and Satou, 2019;Pasini et al., 2006;Roure and Darras, 2016;Shimeld et al., 2005;279 Wagner and Levine, 2012;Wagner et al., 2014), from the Aniseed database (Dardaillon et al., 280 2020) and from the present study, a list of 68 genes with expression in the palps lineage in 281 Ciona and/or Phallusia (Table S3). We plotted the results of our Phallusia RNA-seq data, and 282 found that 70% of the genes were regulated by BMP signaling. Most of them were repressed 283 by BMP, but 20 genes were activated by BMP, and a smaller fraction was repressed or 284 activated depending on the stage. Consequently, the precise function of BMP that is likely to 285 be dynamic in the course of palps differentiation needs to be further investigated in details. 286 Interestingly, Notch is likely to play a role in the specification of the different cell types that 287 compose the palps. For instance, it has been shown that activating Notch represses palps 288 neuronal markers in H. roretzi (Akanuma et al., 2002). We found 30 genes regulated by 289 Notch in our dataset. 290 291 292

Discussion 293
We have shown that BMP signaling regulates two distinct steps of palps formation in C. 294 intestinalis: ANB specification and papilla vs inter-papilla specification. Moreover, we have 295 shown conservation of gene expression and regulation by BMP in P. mammillata. 296 297 Signaling pathway inhibition and ANB specification 298 ANB specification is regulated by inputs from several signaling pathways: FGF, Wnt and BMP. 299 While FGF is positively required early on, at the time of neural induction (32-cell stage), all 3 300 pathways are inactive at the time of ANB fate acquisition as revealed by the expression of 301 Foxc (mid-gastrula). This situation is reminiscent of data from vertebrates where anterior 302 neural fate is determined by the triple inhibition of BMP, Nodal and Wnt pathways 303 (Andoniadou and Martinez-Barbera, 2013;Niehrs et al., 2003;Wilson and Houart, 2004). It 304 would thus be interesting to test the function of Nodal inhibition in ANB specification since 305 we have already shown that it is involved in posterior neural fate determination in Ciona 306 (Roure et al., 2014). While it appears that active FGF, Wnt or BMP signaling is incompatible 307 with ANB determination, the specific function of each pathway seems different. FGF appears 308 to regulate anterior CNS vs ANB fate decision along the antero-posterior axis (Wagner and 309 Levine, 2012). Wnt seems to regulate Foxc+ ANB fate vs Foxc-ANB fate along the medio-310 lateral/dorso-ventral axis (Feinberg et al., 2019). Finally, BMP might participate in the 311 segregation between ANB and immediately anterior/ventral epidermal fates. Finer details on 312 the function of these pathway in ANB fate determination and on their likely cross-talk should 313 be an exciting line of research in this simple and geometric model system. 314

From ANB to palps differentiation 316
Our results of late inhibition of BMP signaling (from gastrula stages) indicate that Foxg, 317 expressed in a single row of cells with a U-shape, delineates cells competent to become 318 papilla. A network of gene interactions has previously been identified that regulates the 319 transition of Foxg from a U-shape to 3-spots eventually forming protruding papillae (Liu and 320 Satou, 2019). BMP is an input to this network, presumably through the regulation of 321 Sp6/7/8/9 expression. Importantly, we have shown that BMP signaling is active in the 322 median palps forming region before the onset of Foxg and Sp6/7/8/9 expression. This region 323 most likely corresponds to the future ventral palp. However, activating BMP at this stage, 324 (Akanuma et al., 2002;Coulcher et al., 2020;Joyce Tang et al., 2013;Pasini et al., 2006;357 Roure and Darras, 2016 Biologiques Marines in Banyuls-sur-mer (EMBRC-France) following diving or by professional 369 fishermen following trawling in the Banyuls-sur-mer (France) area. Gametes collection, in 370 vitro fertilization, dechorionation and electroporation were performed as previously 371 described (Coulcher et al., 2020;Darras, 2021); and staging of embryos was performed 372 according to the developmental table of Ciona robusta (Hotta et al., 2007). 373 Electroporation constructs used in this study have been previously described (Pasini et al., 374 2006 (Racioppi et al., 2014). Briefly, 394 digoxigenin-labeled probes were recognized using an anti-DIG antibody coupled to 395 peroxidase (11207733910, Roche), and fluorescein-labeled probes were recognized using an 396 anti-FLUO antibody coupled to peroxidase (11426346910, Roche). Fluorescence signal was 397 produced using the TSA plus kit (NEL753001KT, Perkin-Elmer) following manufacturer's 398 recommendations with cyanin3 and fluorescein for DIG-and FLUO-probes respectively. 399 Active BMP signaling was visualized by immunostaining using a rabbit monoclonal antibody 400 against mammal Smad1, Smad5 and Smad8 phosphorylated at two serine residues at the C-401 terminal end (clone 41D10, #9516, Cell Signaling Technology) diluted at 1:200. The epitope is 402 present in the single ortholog Smad1/5/8 of both Ciona intestinalis and Phallusia 403 mammillata. Anti-rabbit coupled to Alexa Fluor 568 (A11011, Invitrogen) was used at 1:400 404 for visualization. Similar data were obtained using another antibody (clone D5B10, #13820, 405 Cell Signaling Technology) (data not shown). Membranes were stained using Alexa Fluor 594 406 phalloidin (A12381, Invitrogen) used at 1:1000. Nuclei were stained using DAPI. Image 407 acquisition was performed using confocal microscopy (Leica SP8-X, BioPiC platform, Banyuls-408 sur-mer). Confocal z-stacks were visualized and analyzed in 3D using the Imaris 8.3 software 409 (Bitplane). In particular, this software was used to count the number of cells expressing a 410 gene of interest. In brief, fluorescent signals were converted as 3D objects: in situ 411 hybridization signals as surface objects, and DAPI-labeled nuclei as spots. The number of 412 spots within a given surface was used as a proxy for the number of cells expressing a gene. 413 Snapshots of such analyses and 3D renderings are shown in Fig 1, 3 Figure S1   None LGALS9C