bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Annelids win again: the first evidence of Hox antisense transcription in Spiralia.

2 Elena L. Novikova*, Nadezhda I. Bakalenko and Milana A. Kulakova*

3 St. Petersburg State University, St. Petersburg, 199034 Russia

4 *Corresponding authors:

5 ELN: [email protected]

6 MAK: [email protected]

7

8 Abstract

9 To date it is becoming more and more obvious that multiple non-coding RNAs, once considered 10 to be transcriptional noise, play a huge role in gene regulation during animal ontogenesis. Hox 11 genes are key regulators of embryonic development, growth and regeneration of all bilaterian 12 animals. It was shown that mammalian Hox loci are transcribed in both directions and non- 13 coding RNAs maintain and control the normal functioning of Hox clusters. We revealed 14 antisense transcripts of most of Hox genes in two lophotrochozoans, errant annelids Alitta virens 15 and Platynereis dumerilii. It is for the first time when non-coding RNAs associated with Hox 16 genes are found in spiralian animals. All these asRNAs can be referred to as natural antisense 17 transcripts (NATs). We analyzed the expression of all detected NATs using sense probes to their 18 Hox mRNAs during larval and postlarval development and regeneration by whole mount in situ 19 hybridization (WMISH). We managed to clone several asRNAs (Avi-antiHox4-1, Avi-antiHox4- 20 2 and Avi-antiHox5) of these annelids and analyzed their expression patterns as well. Our data 21 indicate variable and complicated interplay between sense and antisense Hox transcripts during 22 development and growth of two annelids. The presence of Hox antisense transcription in the 23 representatives of different bilaterian clades (mammals, myriapods and annelids) and similar 24 expression relationships in sense-antisense pairs suggest that this can be the ancestral feature of 25 Hox cluster regulation.

26 Key words: Annelids, Antisense RNA, Hox genes, lncRNA, Lophotrochozoa, NATs

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29 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

30 Introduction

31 Eukaryotic genome mostly consists of non-coding sequences. About two thirds of the 32 mammalian genome is actively transcribed but less than 2% of genomic DNA is protein-coding 33 (Taft et al., 2007; Djebali et al., 2012). Among the plethora of non-coding regulatory transcripts 34 such as microRNAs, PIWI-interacting RNAs, small nucleolar RNAs, and small interfering 35 RNAs (Horabin, 2013; reviewed in Hombach and Kretz, 2016), an essential part is occupied by 36 long non-coding RNAs (lncRNA). They are defined as non-coding transcripts that are more than 37 200 nt long and lack a long open reading frame (Brosnan and Voinnet, 2009). LncRNAs contain 38 fewer exons than mRNAs and demonstrate a low expression level across the tissues (Derrien et 39 al., 2012). Most of the known lncRNAs are transcribed with the help of the same transcription 40 machinery as mRNAs, i.e. RNA-polymerase II, have a 5′ terminal methylguanosine cap and are 41 generally spliced and polyadenylated (Ponting and Belgard, 2010). According to their position 42 relative to protein-coding genes, lncRNAs can be intergenic (lincRNAs), intronic, that is, coded 43 in gene introns, enhancer-associated (eRNAs) or transcribed from the same promoter as a 44 protein-coding gene (pancRNA). They can also be transcribed from the strand lying opposite to 45 the protein-coding one and overlapping with the coding sequence (He et al., 2008; Ponting et al., 46 2009; reviewed in Schmitz et al., 2016). The latter group of lncRNAs is referred to as Natural 47 Antisense Transcripts (NATs) (Moran et al., 2012). According to some estimations, up to 70% of 48 mammalian protein-coding genes possess natural antisense counterparts (Chen et al., 2004; 49 Katayama et al., 2005; Faghihi and Wahlestedt, 2009).

50 LncRNAs participate in the regulation of multiple developmental processes such as pausing and 51 release of gene transcription as an adaptation to heat shock during segmentation in Drosophila 52 (Wang et al., 2007), stress response in Arabidopsis thaliana (Liu et al., 2012), sperm formation 53 and the establishment of male identity in Caenorhabditis elegans (Nam and Bartel, 2012), stem 54 cell fate regulation (Guttman et al., 2011), dose compensation (Meller and Rattner, 2002, 55 Froberg et al., 2013) and cardiac development and heart function in mammals (Klattenhoff et al., 56 2013; Grote and Herrmann, 2013). An evidence of the dramatic role of lncRNAs in cancer and 57 metastasis progression has been emerging in recent years (Gupta et al, 2010; Tang et al., 2016; 58 Xie et al., 2016; Leng et al., 2018; Qian et al., 2018; Ghaforui-Fard et al., 2019).

59 The participation of lncRNAs in the control of Hox gene expression during embryonic and adult 60 animal life has been shown in multiple works (Petruk et al., 2006; Rinn et al., 2007; Janssen and 61 Budd, 2010; Wang et al., 2011; Gummalla et al., 2012). The tiling array of Hox genes from 62 human fibroblasts representing 11 distinct positional identities revealed the transcription of 231 63 non-coding RNAs in four Hox loci (Rinn et al., 2007). There is evidence that most of these 64 RNAs are transcribed from the strand opposite to Hox genes. It was shown that 48% of Hox bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

65 lncRNAs displayed an expression pattern along the developmental axis and possessed the 66 sequence motifs specific of ncRNAs located in distal, proximal or posterior sites of the human 67 body (Rinn et al., 2007).

68 Though lncRNAs are abundant in Hox clusters of mammals, the characteristics and function of 69 only a few of them have been described. Among them there are three lncRNAs from HoxA 70 cluster: HOTAIRM1 (HOX antisense intergenic RNA myeloid 1), which is located between 71 human HOXA1 and HOXA2 genes and transcribed antisense to HOXA genes (Zhang et al., 72 2009), HOXA‐AS2 localized between HOXA3 and HOXA4 (Zhao et al., 2013) and lincRNA 73 HOTTIP localized upstream from HOXA13 (Pradeepa et al., 2017). Long non-coding RNA 74 HOTAIR (HOX Antisense Intergenic RNA) is localized between НOXC11 and HOXC12 genes 75 and participates in the trans-regulation of genes from НОХD locus (Li et al., 2013). All the 76 lncRNAs mentioned above are known to regulate various oncogenic processes such as breast 77 cancer, esophageal cancer, lung cancer and gastric cancer (reviewed in Yu and Li, 2015; 78 reviewed in Botti et al., 2018). A study of caecum development in herbivores and omnivores 79 revealed the existence of two non-coding RNAs, named Hotdog (Hog) and Twin of Hotdog 80 (Tog), which are located in a gene desert flanking the HoxD cluster. These RNAs share a 81 common transcription start site and are specifically transcribed from opposite DNA strands in the 82 growing caecum in midgestation (Delpretti et al., 2013).

83 It has been suggested that an intensive antisense and polycistronic transcription across 84 mammalian Hox clusters may be the reason why Hox genes are packed close together in the 85 mammalian clusters (Mainguy et al., 2007). These clusters are indeed relatively short and 86 compact.

87 There are also several examples of antisense transcription within Hox clusters in the Ecdysozoan 88 clade (Brena et al., 2006; Petruk et al., 2006; Janssen and Budd, 2010; Gummalla et al., 2012). 89 Drosophila lncRNA bxd is transcribed from the area between ultrabithorax (Ubx) and 90 abdominal-A (Abd-A) genes. It controls the Ubx expression through transcriptional interference, 91 a mechanism ensuring down-regulation of 3’ genes by transcription of upstream ncRNAs 92 through their promoters (Petruk et al., 2006; Pease et al., 2013). The same mechanism of gene 93 regulation is at work in the 92-kb transcript infraabdominal-8 (iab-8), which is transcribed from 94 the iab region localized between Abd-A and Abd-B genes (Gummalla et al., 2012). An antisense 95 Ubx transcript is expressed in the development of myriapods Strigamia maritima, Glomeris 96 marginata and Lithobius forficatus (Brena et al., 2006; Janssen and Budd, 2010).

97 The presence of antisense transcription within Hox clusters in the representatives of two different 98 clades brings up a question of whether this is a plesiomorphic bilaterian feature or an invention bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

99 of mammals serving to support the integrity of their compact Hox clusters. To answer this 100 question, an evidence from the third clade, the Lophotrochozoa, is strongly needed. At present, 101 there are no data about lncRNA Hox expression, particularly antisense RNA (asRNA) 102 transcription, in this clade.

103 There are two options to detect antisense transcription by whole mount in situ hybridization 104 (WMISH). In case the sequence of antisense transcript significantly overlap the sense one (by 105 more than 500 bp) we can use sense probe to mRNA sequence to reveal antisense transcription. 106 The second option is to clone the antisense fragment and use the probe (antisense probe) to this 107 fragment to detect antisense transcription in WMISH. In this study, we revealed antisense 108 transcripts of most of the Hox genes in two lophotrochozoans, errant polychaetes Alitta virens 109 and Platynereis dumerilii. We managed to clone several asRNAs (Avi-antiHox4-1, Avi- 110 antiHox4-2 and Avi-antiHox5) of these annelids and analyzed their expression during larval and 111 postlarval development and regeneration by WMISH.

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125 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

126 Results

127 Cloning of sense and antisense Hox transcripts

128 Cloning of sense and antisense Hox transcripts of A. virens and P. dumerilii was mostly 129 performed in the Laboratory for Development and Evolution, Department of Zoology, University 130 of Cambridge). The search and cloning of rare low-abundant RNAs is technically complicated. 131 We used the RACE method (Rapid Amplification of cDNA Ends) which allowed us to obtain 3'- 132 and 5'- ends of target RNAs. Since we observed the localization of all antisense transcripts 133 detected by sense probes to mRNA sequences in the cytoplasm we suggested that these 134 transcripts are polyadenylated from 3’-end. Thus, we performed amplification of transcript 3’- 135 end using the primer, which contained polyT sequence. This sequence is complementary to 136 polyA on the 3’-end of target transcript. For the amplification of 5’-ends SMARTTM (Switching 137 Mechanism At 5' end of the RNA Transcript) RACE was used (Clontech).

138 For primer construction we used the known sequences of sense Hox gene fragments. We 139 constructed three pairs of forward and reverse primers for different sites of each Hox sequence. 140 The primers’ length varied from 23 to 28 nt with Tm around 70 0C. The cloned sequences were 141 ligated into pGEM®-T Easy Vector (Promega).

142 As a result the following antisense transcripts were cloned (Table S1):

143 1) a 3’ fragment of Avi-antiHox5 (Gene Bank KP100547). This fragment is 938 nt long and 144 complementary to the 5’-end of Avi-Hox5 mRNA and 5’UTR.

145 2) a 3’ fragment of Avi-antiHox4 (Avi-antiHox4_1; Gene Bank KX998894.1). This fragment is 146 1212 nt long and complementary to the 3’-exon of Avi-Hox4 mRNA (Fig. 1).

147 3) a 3’ fragment of Avi-antiHox4 (Avi-antiHox4_2; Gene Bank KX998895.1). This fragment is 148 739 nt long and includes a fragment of the 5’ intergenic region (Fig. 1).

149 4) a 5’ fragment of Pdum-antiHox7 which is complementary to the 3’-end of Pdum-Hox7 and 150 464 nt long.

151 5) a 5’ fragment of Avi-antiHox7 which is complementary to the 3’-end of Avi-Hox7 and 1244 nt 152 long.

153

154 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

155 In situ hybridization

156 We have previously published a detailed description of the expression of coding transcripts from 157 the Avi-Hox genes during development and regeneration of A. virens (Kulakova et al., 2007, 158 Bakalenko et al., 2013, Novikova et al., 2013). Therefore, here we present these sense patterns 159 only for the sake of comparison with the antisense expression patterns. Such comparisons are 160 essential for understanding the transcription dynamics and the possible roles of the antisense 161 transcripts.

162 A note on Terminology

163 We use the term “antisense RNA probe” to mean a probe containing the sequence which is 164 antisense with respect to the coding strand of the Hox gene, and which therefore hybridises to 165 and detects transcripts from the strand that encodes the Hox protein. Conversely, the term “sense 166 RNA probe” is used to mean a probe that contains sequences from the sense (i.e. Hox protein 167 encoding) sequence of the gene. Such probes will hybridise to antisense transcripts derived from 168 the same genomic region.

169 antiHox1

170 To detect antisense transcription from the region of the Avi-Hox1 gene, we used a digoxigenin- 171 labelled A. virens sense RNA probe (548 bp) (Fig. 2 A) that overlapped the homeobox (HB) and 172 3’UTR of Avi-Hox1. Avi-antiHox1 is expressed in segment ectoderm, the coelom and the gut in 173 the posterior quarter of the body of juvenile and regenerating worms. Weak expression is visible 174 in pygidial cirri and the esophageal region (Fig. 2 C (a, b, c); B, orange color). A probe 175 containing the antisense strand of this same sequence, complementary to mRNA, clearly reveals 176 sense transcription in the ganglia of young but already formed segments, in the cirri and in the 177 esophageal area (Fig. 2 D (a, b, c); B, green color). It is noteworthy that the domain of maximal 178 expression of Avi-antiHox1 coincides with one of the zones of minimal Avi-Hox1 transcription 179 (Fig. 2 C (b), D (b)). However, there are areas (esophagus, pygidial cirri) where both transcripts 180 are visible.

181 antiHox2

182 Similarly to antiHox1, the transcription of antisense Hox2 of A. virens was revealed with sense 183 RNA probe (580 bp) synthesized from 3’- area of coding Avi-Hox2 sequence (Fig. 3 A). Avi- 184 antiHox2 transcript is detected in the semicircle of the ectodermal cells on the dorsal site of 185 prepygidial area and in the esophagus (Fig. 3 C (a, b, c); B, orange color). The first expression 186 domain probably corresponds to the ectodermal growth zone. The second domain coincides with bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

187 the territory of Avi-antiHox1 expression (Fig. 2 C (a); B, orange color). Avi-Hox2 mRNA 188 transcripts are visible in the mesoderm of the youngest segments, presumably, in the mesodermal 189 growth zone and at the base of the jaws (Fig. 3 D (a, b, c); B, green color). It is interesting that 190 the expression zones of sense and antisense Avi-Hox2 transcripts do not overlap but adjoin each 191 other.

192 antiHox3

193 We failed to detect an antisense transcription of A. virens Hox3 with the sense RNA probe (550 194 nt) corresponding to the second exon and 3’UTR of Avi-Hox3 mRNA. However, a weak 195 antisense transcription of Hox3 is visible in P. dumerilii. To reveal it, we used sense RNA probe 196 (619 nt) that overlaps the first exon without 5’UTR and partially the second exon (Fig. 4 A). 197 Pdum-antiHox3 expression is detected only at the nectochaete stage in the forming esophagus 198 (Fig. 4 B (a)). We cannot be sure that antisense RNA of this gene is transcribed in such a narrow 199 frame. This result can be due to the fact that we detect only the maximal expression, which 200 occurs at the nectochaete stage. mRNA of Pdum-Hox3 marks the territory of the future growth 201 zone from the metatrochophore stage, and this expression is retained in juvenile worms (Fig. 4 B 202 (b)).

203 antiHox4

204 To analyze the antisense transcription of A. virens Hox4, we used an antisense RNA probe for 205 the largest of the cloned fragments, Avi-antiHox4_1 (1212 nt), and a sense RNA probe for 3’- 206 fragment of Avi-Hox4 mRNA (618 nt) (Fig. 5 A). Avi-antiHox4_1 is detected for the first time at 207 the early trochophore stage at 50 h. The expression domain is represented by paired dots in the 208 nuclei of mesodermal band cells (Fig. 5 D (b)). In four hours the antisense RNA is revealed in 209 the nuclei of adjacent ectodermal cells that will contribute to the formation of the third larval 210 segment and the pygidium (Fig. 5 D (c)). Four hours later Avi-antiHox4_1 transcription starts in 211 the cytoplasm (Fig. 5 D (d)). The detected signal gradually loses the nuclear localization and by 212 the late trochophore stage is retained only in the cytoplasm of the ectodermal cells (Fig. 5 D (e, 213 f)). At the metatrochophore stage (120 h) the transcript is revealed in the area of the future 214 pygidium and in a few surface cells, localized along the midline of elongating vegetal plate (Fig. 215 5 D (g)). Avi-antiHox4_1 expression persists in the pygidium, pygidial cirri and ectodermal 216 growth zone at the nectochaete stage (Fig. 5 D (h)).

217 It is interesting to compare the distribution of Avi-antiHox4_1 with the pattern of mRNA 218 expression of Avi-Hox4. This gene works on the territory of the second and the third larval 219 chaetiger with an early expression initiation at 46 hpf (hour post fertilization). Its transcript is 220 detected in the mesodermal bands and larval ectoderm (Fig. 5 E (a-h)). Previously we described bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

221 in detail the mesodermal expression of A. virens Hox genes (Kulakova et al., 2017). The sense 222 transcript of Avi-Hox4 appears four hours after the antisense one and demonstrates only 223 cytoplasmic localization. Before the metatrochophore stage the territories of sense and antisense 224 transcription partially overlap at the level of the posterior boundary of Avi-Hox4 domain and the 225 anterior boundary of Avi-antiHox4_1 domain. Later the areas of their transcription overlap in 226 pygidial cirri (Fig. 6 A (a, e)). We failed to detect the expression pattern of the smaller of the two 227 cloned antisense transcripts (Avi-antiHox4_2, 739 nt) (Fig. 1).

228 Using the sense RNA probe covering the second exon, we revealed one more antisense transcript 229 with a different expression pattern than that described above. This transcript is detected at the 230 trochophore stage at the margins of the closing vegetal plate (Fig. 5 B (a)). Noteworthy, there is 231 no sense transcription in the ectoderm at this stage, and the cloned antisense transcript (Avi- 232 antiHox4_1) is detected in the mesodermal cells (Fig. 5 D (b), E (b); С (a)). At the early 233 metatrochophore stage the sense RNA probe is visible in the surface cells of the ventral part of 234 the vegetal plate in all the three larval segments. Sense and antisense transcription domains 235 partially overlap at this stage (Fig. 5 B (b), C (b)). Later, the ectodermal expression Avi-antiHox4 236 (non-cloned) vanishes, and by the late metatrochophore stage the signal is detected only in deep, 237 presumably mesodermal cells at the basis of the pygidial anlagen. During this period, the sense 238 transcript is localized in the segmental ectoderm (Fig. 5 B (c), C (c)).

239 In juvenile worms Avi-antiHox4_1 is detected only in the ectodermal growth zone and in the 240 pygidium and cirri (Fig. 6 A (a, b); B, purple color). The non-cloned antisense transcript that 241 was detected by the sense RNA probe is localized in the mesoderm of the young segments (Fig. 242 6 A (c, d); B, orange color). Avi-Hox4 mRNA marks the ectoderm of young segments, the 243 ganglia of the ventral nerve cord and individual cells in pygidial cirri (Fig. 6 A (e, f); B green 244 color). Thus, in juvenile worms the sense transcript and both antisense ones are localized in a 245 complementary manner in non-overlapping zones. The only area where sense and antisense 246 transcripts seem to overlap is the pygidial cirri. However, since Avi-antiHox4_1 demonstrates a 247 non-homogeneous distribution and the sense RNA is detected in separate cells, we can assume 248 that their localization in pygidial cirri is also complementary. Double WMISH is necessary to 249 ascertain the co-localization of these transcripts.

250 Using the sense RNA probe (592 nt) (Fig. 7 A), we managed to detect an antisense transcript of 251 Hox4 gene of P. dumerilii (Fig. 7 B (a, b, c)). We determined the localization of this transcript in 252 the ectoderm. At the trochophore stage its expression pattern partially overlaps with the 253 transcription zone of the sense RNA (Fig. 7 B (a), C (a)), but the situation changes dramatically 254 by the nectochaete stage. The sense transcript is clearly visible in the ectoderm of the second and bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

255 the third segments, while the antisense transcript is detected in the pygidium, the stomodeum and 256 in a few cells of the head (Fig. 7 B (b), C (b)).

257 Noteworthy, Avi-Hox4 antisense RNA is not detected in the mouth and in the episphere (Fig. 5 D 258 (h)). Later, Pdum-antiHox4 is transcribed only in the pygidium, strictly outside the territory of 259 Pdum-Hox4 mRNA expression (Fig. 7 B (c), C (c)).

260 Pdum-antiHox4 transcription pattern reminds in some respects that of Avi-antiHox4_1, but we 261 cannot consider these transcripts as homologous. Firstly, Pdum-antiHox4 is transcribed from the 262 sequence of the first and the second exons, while Avi-antiHox4_1 does not include the first exon 263 (Fig. 1). Secondly, even if we assume that the Pdum-Hox4 sense RNA probe reveals Pdum- 264 antiHox4 only at the level of the second exon, a methodological contradiction arises: the part of 265 the probe including the second exon is too short (127 nt) for the transcript to be revealed by 266 WMISH. 267

268 antiHox5

269 Previously we described the transcription pattern of Avi-Hox5 asRNA revealed by the sense 270 RNA probe (1030 nt) in juvenile worms during normal growth and regeneration (Bakalenko et 271 al., 2013; Novikova et al., 2013). The cloned fragment of Avi-Hox5 mRNA used for the 272 synthesis of sense and antisense probes includes a part of the first exon and the complete protein- 273 coding sequence of the second exon with a short 3’UTR area (Fig. 8 A). We managed to clone a 274 fragment of asRNA of Avi-Hox5 gene, which is complementary to 5’UTR of the coding 275 transcript, and a small protein-coding area, which overlaps the activator domain (Fig. 8 A). We 276 analyzed the transcription pattern of the cloned Avi-antiHox5 (GenBank: KP100547.1) and 277 compared it to the pattern of the protein-coding sequence and the sense RNA probe pattern.

278 Both probes to antisense RNAs demonstrate similar transcription dynamics and patterns. Here 279 we describe the early expression of the cloned fragment because it was studied in more detail 280 (Fig. 8 B (a-p), C). The antisense transcription starts before the sense one. Avi-Hox5 mRNA is 281 first detected at the late trochophore stage (Fig. 8 D (f)), while both antisense transcripts are first 282 visible almost 50 hours earlier at the early trochophore stage (Fig. 8 B (a, b) (shown for cloned 283 asRNA). The antisense expression starts in the bilaterally symmetrical groups of ectodermal cells 284 localized close to the vegetal pole of the larva (Fig. 8 B (a - f)). Initially asRNA is detected only 285 in the nuclei (52 hpf), entering the cytoplasm in about 6 hours (58 hpf) (Fig. 8 C). The number of 286 positive cells rapidly increases, and they form a ring by the middle trochophore stage (60-80 hpf; 287 Fig. 8 B (g - p)). bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

288 Noteworthy, Avi-antiHox5 retains the nuclear localization starting from very early stages (52-56 289 hpf) and to the middle trochophore stage (80 hpf). Similar to Avi-antiHox4_1 (Fig. 5 D (b, c)), 290 this transcript forms paired conglomerates in the nucleus (Fig. 8 C). This localization is probably 291 due to the activity of two allelic loci in the diploid genome.

292 Since the transcription pattern of the cloned Avi-antiHox5 and the sense RNA probe pattern were 293 very similar, we must have detected the same antisense RNA with the probes to its 3’ and 5’ 294 ends. Fig. 8 D demonstrates the larval expression, as revealed with the sense RNA probe (Fig. 8 295 D (a-e)) and as compared to Avi-Hox5 mRNA pattern (Fig. 8 D (f - j)). It can be seen that the 296 territories of antisense and sense RNA transcription overlap. By the late metatrochophore stage 297 (168 hpf) the antisense transcription spreads to the ectoderm of the second segment and is 298 initiated in the mesoderm (dashed line) (Fig. 8 D (d)). By the nectochaete stage the ectodermal 299 expression of antisense RNA gradually vanishes, while the mesodermal transcription in the third 300 segment and future growth zone intensifies (Fig. 8 D (e)). During the same period Avi-Hox5 301 mRNA is intensively expressed in the ectoderm of the third chaetiger of the larva but not in the 302 mesoderm or the growth zone (Fig. 8 D (i, j)).

303 Both antisense transcripts are detected on the territory of the future third larval chaetiger and 304 overlap with the expression pattern of Avi-Lox5 mRNA (described in Kulakova et al., 2007) 305 starting from the middle trochophore stage. We performed double WMISH with the probes to the 306 cloned Avi-antiHox5 (Dig-probe) and Avi-Lox5 (FITC-probe), using BM-purple and Fast Red, 307 respectively, for the signal detection. Based on the results of chromogenic detection, we cannot 308 be sure that the signals are co-localized, but the borders of transcription domains of Avi-antiHox5 309 and Avi-Lox5 coincide (Fig. 9 A).

310 The difference between the expression patterns of Avi-antiHox5 detected with the sense RNA 311 probe and the pattern of the cloned antisense fragment can be seen for the first time at the 312 nectochaete stage (Fig. 9 B). Both antisense transcripts are detected in the growth zone and, less 313 intensively, in the third larval segment. The sense RNA probe signal demonstrates mesodermal 314 localization. Moreover, a weak expression in the pharynx is observed (Fig. 9 B (c, d)). The 315 cloned Avi-antiHox5 transcript is more distinct in the ectoderm (Fig. 9 B (a)). Besides, its 316 expression is initiated in the basis of aciculae (large supporting rods in parapodia) (Fig. 9 B (b)). 317 This is a transient transcription domain, which is not observed in juvenile worms (Fig. 10). Avi- 318 Hox5 mRNA expression maximum is observed in the ganglia of the third larval chaetiger, while 319 both antisense transcripts demonstrate a low expression level there (Fig. 9 B (e, f)).

320 In juvenile worms both antisense transcripts are revealed in the derivatives of all three germ 321 layers and demonstrate a posterior-anterior expression gradient (Fig. 10 A (a - f)). In the nervous bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

322 system the gradients are very short and look complementary to the mRNA gradient (Fig. 10 A 323 (h)). The cloned Avi-antiHox5 fragment is visible in multiple individual cells in the neural 324 system, the surface epithelium, the coelom and the intestine (Fig. 10 A (b, c)). The pattern 325 revealed by sense RNA probe is diffuse and restricted to the ectoderm in a few young segments 326 (Fig. 10 A (e, f)). Avi-Hox5 mRNA is expressed in a wide bidirectional gradient, with the 327 expression maximum localized in the segments of the central part of the postlarval body (Fig. 10 328 A (g)) (Bakalenko et al., 2013). Avi-Hox5 mRNA is detected only in the surface epithelium and 329 in the neural ganglia of the segments that have already formed parapodia. This is clearly visible 330 in optical sections (Fig. 10 A (h, i)). The schematic patterns are presented in Fig. 10 B. Thus, the 331 coding Avi-Hox5 RNA and its antisense transcription seem to work in overlapping territories at 332 the trochophore and the metatrochophore stages and in adjacent territories in the nectochaete and 333 the juvenile worm.

334 The antiHox5 transcription of P. dumerilii is detected with the sense RNA probe, which 335 coincides with the coding mRNA at the level of the first and the second exons excluding the 336 activator domain, 5’UTR and 3’UTR (Fig. 11 A). We analyzed the expression of Pdum- 337 antiHox5 in juvenile worms only and revealed the pattern complementary to the sense one (Fig. 338 11 B, C). The antisense RNA is mainly detected in the ectodermal growth zone, the surface 339 epithelium and in the neural system of the youngest segments. The diffuse signal is similar to the 340 ectodermal pattern of the non-cloned asRNA of A. virens (Fig. 10 A (d, e, f)).

341 antiHox7

342 We have previously described the antisense transcription of the Hox7 gene of Alitta virens 343 during postlarval growth and regeneration (Bakalenko et al., 2013; Novikova et al., 2013). We 344 used the sense RNA probe (522 bp), which coincided with the coding sequence of Avi-Hox7 345 mRNA at the level of the second exon (Fig. 12 A). According to our new data, the antisense 346 transcript starts to express much earlier than the sense one. We first detect it at the late 347 trochophore stage (100 hpf) in the vegetal plate cells (Fig. S1.(a)). Later, the transcript appears in 348 the forming stomodeum and in the neural ectoderm of the third segment of the metatrochophore 349 (Fig. S1 (b)). The antisense RNA is detected in the formed ganglia starting from the third larval 350 segment, and its localization does not coincide with the sense transcription area, as Avi-Hox7 351 mRNA is observed in the growth zone of nectochaetes and juvenile worms at the same time (Fig. 352 12 C (a), D (a)). In older worms the sense and the antisense territories overlap in the nervous 353 system (Fig. 12 C (b, c), D (b, c)). The antisense RNA forms a wide anterior-posterior gradient, 354 with the maximum in the anterior third part of the postlarval body. There is no transcription in 355 the growth zone (Fig. 12 C (b, c), B). The mRNA Avi-Hox7 is revealed in the nervous system 356 and the segment ectoderm in the form of posterior-anterior gradient, the expression in the bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

357 nervous system vanishing in the anterior third part of the body (Fig. 12 D (b, c), B). On the 358 territory where Avi-antiHox7 and Avi-Hox7 patterns overlap, their transcription picture in ganglia 359 is not identical (Fig. 12 C (e), D (e)). Using 5’RACE method, we cloned the fragment of Avi- 360 antiHox7 lncRNA 1244 nt long, which is localized downstream of 3’-area of Avi-Hox7 and 361 overlaps with its 3’UTR (Fig. 12 A, purple color). WMISH with the probe to this fragment did 362 not reveal any transcription.

363 For P. dumerilii we used the sense probe to Pdum-Hox7 mRNA and the probe to the cloned 364 fragment of Pdum-antiHox7. No clear result was identified with any of these probes, perhaps, 365 due to insufficient sensitivity of the method.

366 antiLox4

367 We did not detect the antisense transcription of Avi-Lox4 gene using the sense RNA probe. The 368 size of the probe we used (302 nt) and/or the area of its overlapping with the antisense transcript 369 are probably too small to detect the transcription with WMISH (Table S2).

370 However, we found the antisense transcription of Pdum-Lox4. Sense RNA probe to 3’-end of 371 mRNA corresponds to a homeobox fragment and 3’-UTR (Fig. 13 A). It reveals antiLox4 372 transcription in coeloms of several last segments (Fig. 13 C (a, b), B). The probe to 3’- fragments 373 of the sense RNA detect the expression in neural ganglia and segmental ectoderm in the form of 374 a wide posterior-anterior gradient (Fig. 13 D (a, b), B).

375 antiLox2

376 As in the previous case, we did not reveal the antisense transcription of A. virens Lox2 with the 377 sense RNA probe (516 nt) that corresponds to the 3’-end of the protein-coding sequence and 3’- 378 UTR (Table S2). However, there is an antisense transcript of P. dumerilii Lox2 that was detected 379 with the sense RNA probe (720 nt). This probe coincides with 5’UTR sequence and the coding 380 part of the first and partially second exons (Fig. 14 A). Antisense transcription is detected in the 381 caudal part of the intestine and adjacent coelomic epithelium in the youngest segments (Fig. 14 382 B, C (a, b); ). Pdum-Lox2 mRNA is expressed as a gradient in neural ganglia and in the ectoderm 383 of young segments (Fig. 14 B, D (a, b)). Interestingly, at the early stages of regeneration (10 384 hours post tail amputation) Pdum-antiLox2 starts to express in the neural ganglion close to the 385 amputation site (Fig. 14 C (c, d)). This can be explained either by the changes in the function of 386 the antisense transcript during the regeneration process or, more likely, by an intensification of 387 the neural system expression, which makes it possible to detect the domain invisible during the 388 normal growth. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

389 antiPost2

390 To detect the antisense transcription of Avi-Post2 gene (GenBank: KY020041.1) we used sense 391 RNA probe (706 nt), which coincides with the first exon and a small part of the second one (Fig. 392 15 A). Avi-antiPost2 can be revealed only on larval stages during a short period starting from the 393 middle trochophore stage (70 hpf) and to the early metatrochophore stage (112 hpf) (Fig. 15 B 394 (a-d)). This transcript is visible presumably in the nuclei of the large cells localized on the 395 vegetal pole of the larva (Fig. 15 C (a, b)). Later the descendants of these cells will become a 396 part of the pygidium.

397 At the late trochophore stage the transcript spreads all over the cell, vanishing completely by the 398 middle metatrochophore stage (Fig. 15 B (e, f)). At this time point Avi-Post2 mRNA is 399 synthesized in the region of the future pygidium (Fig. 15 C (d, e)). It is difficult to say if the cells 400 that synthesized the antisense transcript before switch to the synthesis of the sense transcript, 401 since there is a pause between the vanishing of one transcript and the activation of the other. To 402 answer this question, we need to increase the sensitivity of the method and to use more frequent 403 fixations.

404 The antisense transcript of Pdum-Post2 is detected with the sense RNA probe (750 nt), that 405 almost completely coincides with the second exon of this gene (Fig. 16 A). Similarly to Avi- 406 antiPost2, this antisense RNA starts to be synthesized presumably in the nuclei on the territory 407 of the future pygidium long before the activation of the sense transcript. At the early 408 metatrochophore stage, when Pdum-Post2 mRNA expression is initiated in pygidial lobes, the 409 antisense transcript is still detected. It is localized in the adjacent area at the level of the 410 trochoblasts of the telotroch, i.e. around the future pygidial lobes (Fig. 16 B (a), С (a)). This 411 transcription gradually vanishes, but by the stage of late metatrochophore/early nectochaete the 412 new Pdum-antiPost2 expression domain appears in the forming pharynx (Fig. 16 B (b)). Pdum- 413 Post2 mRNA transcription retains in the pygidium (Fig. 16 С (b)). At this stage the sense and 414 antisense transcripts are synthesized not in the adjacent territories but in separate ones, similarly 415 to what we observed in case of sense and antisense Pdum-Hox3 RNA (Fig. 4 B). This may point 416 to the independent function of Pdum-antiPost2 in the development, which is not connected with 417 the control of the transcription and/or translation of the sense RNA. Juvenile worms do not 418 transcribe Pdum-antiPost2 at all (or not on the level detectable by the method we use). However, 419 during regeneration in 48 hours after tail amputation we observe the antisense transcription in the 420 ectoderm of the pygidium anlagen again (Fig. 16 B (c, d)). The sense transcript is detected at this 421 stage mainly in the inner cell mass of regeneration blastema and overlaps a little with the 422 antisense transcription (Fig. 16 С (c, d)). bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

423 Discussion

424 1. Almost all Hox genes of nereidid annelids possess antisense transcripts

425 We investigated the specific transcription patterns of Hox genes’ NATs of nereidid annelids by 426 WMISH using either probes to the cloned antisense RNAs or sense RNA probes to mRNAs. 427 Figure 17 (Fig. 17) summarizes what kinds of probes were used to analyze transcription of 428 different Hox gene RNAs and indicates the sequences used for probe synthesis. Our research 429 shows that despite the difficulties of working with lncRNAs due to their low copy number and a 430 short lifespan, there is a way to study their transcription patterns even without the cloning of the 431 gene fragment. If NATs overlap the coding sequence for more than 500 nt, it is possible to use 432 sense RNA probes for the analysis of the genes of interest (Table S2).

433 We used probes to 5’- and 3’-areas of Hox genes and revealed asRNAs for the genes of all 434 paralogous groups except Lox5 (Table S2). We cannot say with certainty that this Hox gene does 435 not possess asRNA, but the sense RNA probes to the 5’area of Pdum-Lox5 and the 3’-area of 436 Avi-Lox5 did not reveal any expression. All asRNAs of Hox genes, whose patterns were 437 described in this work, can be referred to as NATs (Ariel et al., 2015).

438 The exons of cloned NATs (Avi-antiHox4_1, Avi-antiHox4_2, Avi-antiHox7) consist not only of 439 the coding sequences of Hox genes but also of the intronic and even intergenic regions (Fig. 1). 440 NATs of mammalian Hox genes are organized in a similar way (Mainguy et al., 2007). Besides, 441 we cannot rule out the presence of polycistronic transcripts whose exons overlap with several 442 Hox sequences simultaneously. Such polycistronic sense and antisense RNAs are described for 443 human and murine Hox clusters (Mainguy et al., 2007). In case of in situ screening, RNAs like 444 that should be revealed by the probes to different Hox transcripts but should demonstrate similar 445 expression patterns. We show that all the described patterns are gene-specific, however, asRNAs 446 of several different Hox genes are detected in the same areas, the esophagus and the stomodeum 447 (Fig. 2 B, C (a); Fig.3 B, C (c); Fig.7 B (b); Fig. 16 B (b)).

448 The probes to the cloned antisense sequences and sense RNA probes to the same gene 449 demonstrated variable expression patterns (Fig. 5, 6 (Avi-Hox4); Fig. 9, B (Avi-Hox5)). This 450 means that there are at least three different antisense transcripts for Avi-Hox4 (including Avi- 451 Hox4_2) and two for Avi-Hox5. It is still an open question, how many different asRNAs are 452 transcribed from Hox clusters of nereidids. To answer it, the full sequencing of the clusters and 453 the cap analysis of gene expression (CAGE) are needed.

454 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

455 2. NATs’ patterns of nereidid Hox genes are gene-specific and closely related to sense 456 transcription patterns

457 NATs’ patterns of A. virens and P. dumerilii Hox genes are so variable and dynamic that at first 458 sight it seems impossible to elucidate the common principles of their behavior. To introduce 459 some order into the confusing abundance of data, we decided to consider NATs as possible 460 effectors influencing the functioning of their complementary mRNAs. In this case, we can 461 suggest five possible variants of sense/antisense interaction.

462 a) Complementary transcription on adjacent territories

463 This option is realized for the majority of sense Hox RNAs and their NATs. In juvenile worms 464 and nectochaetes (if this stage was studied) the expression patterns of Avi-antiHox1, Avi- 465 antiHox2, Avi/Pdum-antiHox4, Avi/Pdum-antiHox5, Pdum-antiLox4 и Pdum-antiLox2 are 466 complementary to the sense patterns. This means that the adjacent territories of two transcripts 467 (sense and antisense) do not overlap or overlap on small territories. Avi-Hox2 and Avi-antiHox2 468 are an example of non-overlapping transcription domains. Both transcripts are detected in the 469 growth zone and in the anterior parts of the digestive tract, but sense RNA is localized in the 470 mesodermal component of the growth zone and at the basis of the jaws, while its NAT is located 471 in the ectodermal growth zone and the esophagus (Fig. 3 C, D). Avi/Pdum-Hox5 and Avi/Pdum- 472 antiHox5 transcripts are distributed in the form of opposite gradients and have a narrow conjoint 473 area in the segmental ectoderm and the neural system (Fig. 10, 11).

474 Noteworthy, Avi-antiHox7 is expressed according to this rule at the nectochaete stage and in 475 juvenile worms (4-5 segments), but as the animal grows the areas of sense and antisense 476 expression start to overlap considerably in the neural system due to the activation of sense RNA 477 in postlarval segments (Fig. 12 B, C, D). The expression is probably still complementary but on 478 the level of individual cells, since the pattern in the ganglia differs for the sense and the asRNAs 479 (Fig. 12 C (d), D (d)) (Bakalenko et al., 2013). Pdum-Post2/Pdum-antiPost2 transcripts display 480 complementary transcription only at the stage of the metatrochophore (Fig. 16 B (a), C (a)). 481 Later the complementary transcription is observed only during regeneration (Fig. 16 B (c, d), C 482 (c, d)). Noteworthy, the patterns of different asRNAs of Avi-Hox4 are complementary not only to 483 the sense transcript but also to each other (Fig. 6 A, B).

484 b) Early antisense transcription that precedes and potentially prevents the sense one

485 Transcription of NATs of Avi-Hox5, Avi-Hox7 and Avi/Pdum-Post2 is initiated at the 486 trochophore stage, long before the activation of their sense RNAs. In case of Avi-Hox5 both 487 antisense transcripts precede the expression of mRNA in the third larval segment. Later all the bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

488 three transcripts are co-expressed in this territory until the nectochaete stage (Fig. 8). The early 489 transcription of Avi-antiPost2 precedes the expression of sense RNA at the same domain and on 490 the adjacent territory, but antisense transcription vanishes before mRNA starts to express (Fig. 491 15 B, C (c-e)). Pdum-antiPost2 demonstrates a very similar early pattern, which is retained after 492 the initiation of sense transcription, and two expression areas seem to be strictly complementary 493 at the stage of early metatrochophore (Fig. 16 B (a), C (a)). Avi-antiHox7 transcript appears in 494 larval neuroectoderm at the level of the third segment (Fig. 12 C (a), Fig. S1). Sense 495 transcription never spreads to the territory of the third larval segment, neither at later stages nor 496 even during regeneration, when the anterior boundary of Avi-Hox7 expression domain spreads 497 anteriorly (Novikova et al., 2013).

498 c) Overlap of sense and antisense RNA patterns

499 mRNAs and their NATs of nereidid Hox genes can be temporarily or permanently transcribed at 500 the same territory. The first variant is typical for the larval transcription of Avi/Pdum-antiHox4 501 and Avi-antiHox5 (Fig. 5 B (b), C (b); Fig. 7 B (a), C (a); Fig. 8 D (d), E (d)). Sense and 502 antisense RNAs of Avi-Hox1 permanently overlap in the esophagus (Fig. 2 B, C (a), D (a)).

503 d) Transcription in spatially divided regions

504 In some cases we observe an independent transcription of mRNAs and their NATs on non- 505 adjacent territories: Pdum-Hox3 (growth zone) and Pdum-antiHox3 (esophagus) (Fig. 4 B); 506 Pdum-Hox4 (ectoderm of the second and the third nectochaete segment) and Pdum-antiHox4 507 (stomodeum and distinct cells in the episphere) (Fig. 7 B (b), C (b)); Pdum-Post2 (pygidium) and 508 Pdum-antiPost2 (stomodeum) (Fig. 16 B (b), C (b)). We suggest that in this way NATs prevent a 509 non-specific expression of their mRNAs. Conversely, these observations may indicate an 510 independent function in the regulation of alternative targets, which is usually the case for trans- 511 NATs (Deforges et al., 2019).

512 e) Transcription on the territory of the neighboring Hox gene expression domains

513 The expression patterns of some NATs coincide with the sense patterns of the neighboring Hox 514 genes. For example, Avi-antiHox2 is expressed in the ring of ectodermal cells where Avi-Hox3 515 works (Fig. 3 B (a, b), C (a, b); Fig.4 B (b)) (Bakalenko et al., 2013). At one of the larval stages 516 Avi-antiHox5 and Avi-Lox5 are localized at the same territory of the future third larval segment, 517 which was confirmed by double WMISH (Fig. 9 A). A pattern like this may indicate the 518 presence of bidirectional transcription, which is initiated from the closely localized promoters on 519 the opposite DNA strands. These promoters are under the common regulation, thus the 520 transcribed RNAs are co-expressed (Balbin et al., 2015). A detailed analysis of this observation bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

521 is impossible without the data on the structure of nereidid Hox cluster. It is known that A. virens 522 and P. dumerilii Hox genes are located in one locus but the order of the genes and the presence 523 of inversions in the clusters are unknown (Andreeva et al., 2001; Hui et al., 2012).

524 3. Putative functions of nereidid Hox genes’ NATs and their possible role in development

525 LncRNAs are encoded in the Hox clusters of of mammals, hemichordates, insects and 526 myriapods (Casaca et al., 2018; Mainguy et al., 2007; Freeman et al., 2012; Pettini and 527 Ronshaugen, 2016; Petruk et al., 2006; Brena et al., 2006). These molecules not only control the 528 transcription of Hox genes but also can be involved in the other important processes such as the 529 switch of cellular metabolism from glycolysis to oxidative phosphorylation (Huang et al., 2017). 530 The best studied among them are long intergenic noncoding (linc)RNAs, for example HOTAIR 531 and HOTTIP in mammals (Gupta et al, 2010; Balbin et al., 2015; Li et al., 2019).

532 LncRNAs of nereidid Hox genes belong to another class of molecules. These are NATs (Natural 533 Antisense Transcripts), and they are much more poorly studied in model animals. Human Hox 534 clusters are known to code dozens of NATs (Mainguy et al., 2007), some of which are 535 conservative at least between human and mouse, and so cannot be considered as transcriptional 536 noise (Mainguy et al., 2007; database LncBook: https://bigd.big.ac.cn/lncbook/index). These 537 transcripts overlap Hox genes’ sequences on the regions from 44 nt to 607 nt, and their functions 538 are not yet properly studied. The transcription of some of these ncRNAs is known to be 539 significantly increased or reduced in tumor cells where they co-express with their target mRNAs 540 and stabilize them from degradation, thus supporting the activity of the target gene (Zhang et al., 541 2018). We suggest that some NATs of nereidids’ Hox genes can act using a similar mechanism, 542 since their transcription patterns overlap with the expression domains of the mRNAs and since 543 both transcripts are detected in the cytoplasm.

544 Despite the lack of knowledge concerning the function of antisense transcripts in the Hox cluster, 545 enough data regarding the role of NATs in different regulatory systems have been accumulated. 546 One of the described mechanisms of sense and antisense transcripts’ interplay is transcriptional 547 interference. This mechanism comes into force if sense and antisense transcriptional start points 548 are localized in close proximity to each other. In this case the transcriptional machinery of the 549 antisense strand prevents the synthesis of mRNA by mechanically blocking the polymerase II 550 complex of the sense strand (Latos et al., 2012; Latge et al., 2018; Flippot et al., 2019). 551 Transcriptional interference takes place in cell nuclei. The resulting antisense transcript per se 552 plays no role and eventually degrades. A large number of NATs of nereidids’ Hox genes is 553 indeed detected in the nuclei. This early transcription, which is always larval, usually excludes 554 the synthesis of complementary mRNAs on the same territory (as it was demonstrated for Avi- bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

555 antiHox5 and Avi/Pdum-antiPost2). However, in case of Avi-Hox4/Avi-antiHox4_1 the nuclear 556 asRNA signal accompanies the cytoplasmic signal of mRNA (Fig. 5 D (d), E (d)).

557 The nuclear localization of NATs suggests that there may be other probable mechanisms of 558 transcriptional control. On the one hand, lncRNA molecules may decoy the basic and enhancer- 559 specific transcriptional factors, thus preventing the work of promoters of sense transcripts 560 (Morriss and Cooper, 2017; Flippot et al., 2019). On the other hand, lncRNAs may attract those 561 factors to the promoter and act as guide lncRNAs (Chiu et al., 2018; Flippot et al., 2019). 562 Besides, lncRNAs can function in the nucleus as scaffold molecules (Wang and Chang, 2011). In 563 this case, they recruit to the target site (in cis- or trans-position) not only the individual proteins, 564 but the whole protein complexes of chromatin remodeling, thus changing the chromatin state. 565 Here, as in the previous case, both positive and negative regulation models are possible. It is not 566 implausible that an early (preceding or preventing) expression of NATs of A. virens and P. 567 dumerilii is realized through one of those mechanisms.

568 Finally, NATs can be processed to siRNA (short interfering RNAs). NAT-siRNAs were found in 569 mammals, Drosophila, C. elegans and yeasts (Zhang et al., 2013; Holoch and Moazed, 2015). 570 The mechanism implies the formation of duplexes between lncRNA and the complementary 571 target RNA with further degradation of long dsRNA precursors to the set of small 21-nt 572 molecules by Dicer (Dicer2 in D. melanogaster) ribonuclease. Afterwards the short double- 573 stranded precursors are loaded into RISC-complex and participate in post-transcriptional gene 574 silencing (PTGS). Noteworthy, both the sense and the antisense transcript that form dsRNA are 575 synthesized simultaneously and are processed in the cytoplasm. PTGS in different animals 576 including Drosophila and mammals is closely related to transcriptional gene silencing (TGS) 577 (Pal-Bhadra et al., 2004; Weinberg et al., 2006; Hawkins et al., 2009). It was demonstrated in 578 human cell culture that the antisense small RNA, which is complementary to the promoter region 579 of the target gene, participates in TGS initiation. It is important to note that TGS can be inhibited 580 through the inhibition of RNA polymerase II, which can be a sign of the interaction between 581 siRNA and 5’-region of sense transcript (Weinberg et al., 2006). The mechanisms of PTGS 582 realization in Drosophila and mammals may be at work in annelids as well since in many cases 583 sense and antisense RNAs of nereidids’ Hox genes have been detected in the cytoplasm of the 584 same cells (Avi-Hox4, Avi/Pdum-Hox5). It is still debatable whether our models involves TGS by 585 siRNAs.

586 It is known that transcriptional boundaries of most Hox genes in the postlarval body of nereidids 587 are constantly moving because the worms grow for the most part of their lives. The patterns of 588 many sense/antisense Hox transcripts of A. virens and P. dumerilii retain a dynamic balance. It is 589 important that though the patterns of two transcripts are complementary, the locus that is the bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

590 source of both RNAs (sense and antisense) stays active. This means that the down-regulation of 591 sRNA transcription does not occur through TGS.

592 It is known that Hox activation during regeneration in nereidids takes place approximately in two 593 hours after the injury. This is relatively fast in comparison with amphibians and even planarians 594 (Gardiner et al., 1995; Orii et al., 1999). It is possible that in the postlarval nereidid body the 595 Dicer and Ago “team” functions in the nuclei as it was recently shown for other bilaterian 596 animals (Cernilogar et al., 2011; White et al., 2014; Shuaib et al., 2019). In case the 597 complementary asRNAs are synthesized, these proteins cut Hox gene mRNAs as soon as the 598 latter have been synthesized. Thus, Hox mRNA transcription may be constantly retained but its 599 presence may be controlled by up- and down-regulation of asRNA. This hypothesis clearly 600 explains the fast activation of Hox transcription, but needs to be tested experimentally.

601 It cannot be ruled out that asRNAs of some Hox genes of nereidids serve for maintaining Hox 602 loci in the active state and for preventing heterochromatization. This allows a fast initiation of 603 the sense transcription if the body is damaged. However, if asRNAs serve to maintain an active 604 locus, it is unclear why they should be transported to the cytoplasm.

605 It is worth noting that our probes, regarding their position relative to 3’ or 5’-ends of the target 606 transcript, contain a full or partial sequence of the homeobox. In the case when we managed to 607 clone the fragments of asRNA (Table S1), all of them except for Avi-antiHox5 contained a full or 608 a partial sequence that was complementary to homeobox. This may well be the case for Avi- 609 antiHox5 too, since it was cloned by the 3’RACE method and its 5’-area is unknown. This 610 means that many NATs of nereidids’ Hox genes overlap the most conservative parts of Hox 611 paralogs and potentially may work in trans-mode.

612 All NATs of nereidids’ Hox genes demonstrate complicated transcriptional dynamics. Most of 613 them change their localization from the nuclear to the cytoplasmic in the course of development, 614 but not vice versa. We often observed the change of the interaction character in the 615 sense/antisense pairs. The patterns may overlap at the early developmental stages, becoming 616 complementary later on (Avi-antiHox4_1, Avi-antiHox4 (sense probe) and Avi-antiHox5) and 617 vice versa (Avi-antiHox7). These reversals can indicate the change of NATs’ functions during 618 the development and/or the change of the mechanisms of functional realization.

619 The transcription of Hox genes’ mRNAs in nereidids and an unrelated annelid Capitella sp. 620 (Kulakova et al., 2007; Fröbius et al., 2008; Bakalenko et al., 2013) displays variable patterns 621 during larval and postlarval development. We suggest that this difference, as well as the 622 restricted regeneration capacity of larval segments, is due to the difference in the epigenetic bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

623 tuning of Hox loci in larval and postlarval annelid body. We hypothesize that this tuning may be 624 controlled by NATs.

625 4. The occurrence of NATs of Hox genes among Bilateria: an individual specificity of 626 regulation or an ancestral feature?

627 The first NATs of Hox genes were found and partially cloned in mouse (Hsieh-Li et al., 1995). It 628 was shown that at least four different antisense splice forms are transcribed from Hoxa11 gene 629 and their transcription patterns are complementary to the patterns of the sense transcripts (Hsieh- 630 Li et al., 1995).

631 In the lineage of protostomian animals, NATs associated with Hox cluster (antisense transcripts 632 of Ubx gene (aUbx) were found in three species of myriapods, but not in onychophorans (Brena 633 et al., 2006; Janssen and Budd, 2010; Janssen et al., 2014). Thus, the presence of antisense RNAs 634 was considered as a synapomorphy of Myriapoda (Janssen and Budd, 2010). It should be noted, 635 however, that the structure of onychophoran Hox cluster is yet undescribed, while the cluster of 636 the centipede Strigamia maritima is intact and well-ordered (Chipman et al., 2014). Besides, the 637 Hox cluster of S. maritima is associated with the gene evx/evenskipped similar to the intact Hox 638 clusters of Deuterostomia (Chipman et al., 2014), which indicates a similarity to the ancestral 639 cluster of arthropods. The expression patterns of aUbx are studied in detail in the centipede S. 640 maritima and the millipede Glomeris marginata (Brena et al., 2006; Janssen and Budd, 2010). In 641 general, the principles of transcription of myriapod aUbx are similar to what we observe for 642 some antisense transcripts in nereidids. The Ubx gene is specific for Panarthropoda lineage and 643 its regulation by aUbx transcript is probably limited by the myriapod lineage. However, 644 considering the data on vertebrates (Hsieh-Li et al., 1995; Mainguy et al., 2007) and annelids, we 645 can assume that the principle of NATs’ functioning is not the invention of myriapods but 646 something that was inherited from the regulatory system of the common ancestor of Protostomia 647 and Deuterostomia. Hox gene mRNAs transcription is relatively conservative in A. virens and P. 648 dumerilii (Kulakova et al., 2007), but the expression patterns of NATs of orthologous Hox genes 649 of nereidids demonstrate only a remote resemblance (Avi-antiPost2 and Pdum-antiPost; Avi- 650 antiHox4 and Pdum-antiHox4; Avi-antiHox5 and Pdum-antiHox5). Since there is a probability 651 that our probes reveal non-homologous asRNAs, we should assert the evolutionary lability of 652 NATs with a certain caution. In general, most of lncRNAs are much less conservative than 653 mRNAs or miRNAs (Jarroux et al., 2017). NATs transcribed from Hox clusters of vertebrates, 654 myriapods and annelids can contain the conservative parts in the zones overlapping with protein- 655 coding sequences, especially if this sequence is homeobox. However, this does not imply a 656 structural or functional conservatism of these lncRNAs. It is more likely that bilateral animals 657 with intact Hox clusters use a similar principle of regulation, which mechanism is yet obscure. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

658 We may expect animals with a disorganized or an atomized cluster to possess a poor repertoire 659 of Hox-associated lncRNAs. For example, the topological homologue of Hotairm1 was not 660 found in the ascidian Ciona intestinalis although it exists in tetrapods, teleosts and even in 661 amphioxus (Herrera-Úbeda et al., 2019). In Drosophila, whose cluster is disorganized, although 662 to a lesser extent, the number of Hox lncRNAs is significantly reduced compared to vertebrates. 663 Unfortunately, this hypothesis remains a raw guess, because the data obtained on the model 664 objects are patently insufficient for testing it.

665 Conclusion

666 Bilateral animals possess a rich repertoire of variable mechanisms for controlling the functioning 667 of Hox clusters. Many of these mechanisms are lineage-specific, such as the system of cis- 668 regulatory modules, which integrate the incoming signals from Gap proteins and segmentation 669 proteins during insect development. Mammals use lincRNAs (HOTAIR, HOTTIP, etc.), which 670 are absent in protostomes. However, both insects and vertebrates possess Hox regulation by 671 single specific enhancers or intergenic lncRNAs, which implies the plesiomorphy of this 672 regulatory principle. We suggest that NATs of Hox genes found in mammals, myriapods and 673 annelids are an example of such an ancestral regulatory mechanism, with individual elements 674 having evolved independently in different bilaterian lineages. Hox NATs system is still poorly 675 understood, and the nereidid annelids seem to be a good model for studies of this kind. In these 676 animals NATs of almost all Hox genes can be easily found and analyzed. Further comparative 677 analysis of Hox NATs functioning in the representatives of three evolutionary distinct clades is 678 needed for the reconstruction of the regulatory evolution of Hox clusters.

679 Materials and methods

680 Animals

681 Adult Alitta virens were collected in the Chupa Inlet, the White Sea, near the “Kartesh” marine 682 research station of the Zoological Institute of the Russian Academy of Sciences. Mature animals 683 were caught with a hand net near the water surface during the spawning period (June and July). 684 Artificial fertilization and cultivation of the embryos were carried out at 10.5°C. The culture of 685 postlarval animals was kept in the laboratory of experimental embryology (Petergof, Russia) 0 686 under the following conditions: temperature, 18°C; salinity, 23 /00; artificial sea water (Red Sea 687 salt). The culture of Platynereis dumerilii is kept in the laboratory of experimental embryology at 688 the same temperature, salinity 23 ‰; artificial sea water (Red Sea salt).

689 Regeneration experiment bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

690 Juvenile worms consisting of 20-30 segments (A. virens) and 15-20 segments (P. dumerilii) were 691 relaxed in clove oil (Sigma) of a low concentration for 5 min and cut into two pieces. 692 Experimental details are provided in Novikova et al., 2013.

693 Gene cloning

694 Gene cloning was performed in the Laboratory for Development and Evolution, Department of 695 Zoology, University of Cambridge. Total RNA was isolated from pygidia and several posterior 696 segments of young regenerates (7-10 days of regeneration) of Alitta virens and Platynereis 697 dumerilii using TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. To 698 obtain 3’ and 5’ ends of transcripts of Avi-antiHox4, Avi-antiHox5, Pdum-Lox4, Pdum-Lox2 and 699 Avi-Post2 the SMARTTM (Switching Mechanism At 5' end of the RNA Transcript) RACE 700 method (SMARTTM RACE cDNA amplification kit, Clontech) was used (Brena et al., 2006). To 701 design RACE primers, the sequences of Hox genes of A. virens and P.dumerilii described earlier 702 were used (Andreeva et al., 2001). Several forward and reverse primers for each Hox sequence 703 were generated. To obtain 3’-ends of antisense (as) transcripts and 5’-ends of sense transcripts, 704 reverse primers were used. 5’-ends of antisense transcripts and 3’-ends of sense transcripts were 705 obtained with forward primers. In this paper, we denote the described or mentioned sequences 706 with the help of abbreviations currently used in GenBank for the studied animals: Avi for Alitta 707 virens and Pdum for Platynereis dumerilii. However, many genes of these animals, including a 708 few antisense ncRNAs described in this work, are registered in GenBank under old designations: 709 Nvi for Alitta virens (from the former species name Nereis virens) and Pdu for Platynereis 710 dumerilii. GenBank accession numbers for the cDNA sequences are as follows: Avi-antiHox4_1 711 - KX998894.1; Avi-antiHox4_2 - KX998895.1; Avi-antiHox5 - KP100547.1.

712 Whole mount in situ hybridization (WMISH) and double WMISH

713 We used similar WMISH protocols for A. virens and for P dumerilii. For details of WMISH of 714 larval stages see Kulakova et al., 2007, of postlarval stages, see Bakalenko et al., 2013, of 715 regeneration, see Novikova et al., 2013. Minor modifications applied to the previously developed 716 WMISH protocols for the detection of antisense transcripts were as follows: increasing the time 717 and the number of washings after incubation with the probe and the antibodies, increasing the 718 temperature of washings up to 68⁰ C after the probe incubation, increasing the time of BM- 719 Purple (Roche) incubation up to 48 hours. The detailed protocols are available upon request. 720 Double WMISH was performed using two substrates: BM-Purple and Fast Red RC (Sigma 721 Aldrich). For double WMISH probe synthesis, we used DIG RNA Labeling Mix (for BM-Purple 722 detection) and Fluorescein RNA Labeling Mix (for Fast Red detection).

723 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

724 Acknowledgements

725 The authors are grateful to Professor Michael Akam for providing the opportunity to N.B. to 726 perform cloning in his Laboratory for Development and Evolution, Department of Zoology, 727 University of Cambridge and to Carlo Brena for priceless methodical recommendations and 728 support in cloning Hox antisense transcripts. We thank the staff of the White Sea Biological 729 Station “Kartesh” (Zoological Institute, Russian Academy of Science) for help in collecting and 730 maintaining A. virens. The research was performed using the facilities of the Research park of 731 Saint Petersburg State University “CHROMAS” and “Culture Collection of Microorganisms”.

732 Competing interests

733 The authors declare no competing or financial interests.

734 Author Contributions

735 N.I.B. performed cloning. E.L.N., M.A.K. and N.I.B. performed WMISH. E.L.N. and M.A.K. 736 conceptualized the project, analyzed the results, interpreted the results and wrote the manuscript. 737 M.A.K. supervised the project. E.L.N. performed regeneration experiments and acquired funding 738 (18-04-00450), M.A.K. participated in grant research (19-14-00346).

739 Funding

740 This research is supported by RFBR grant 18-04-00450 to ELN (WMISH reagents), RSCF grant 741 19-14-00346 (reagents for RNA extraction and salary) and EMBO short-term fellowship ASTF 742 143-2014.

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753 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

754 References:

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913 57. Pal-Bhadra, M., Leibovitch, B.A., Gandhi, S.G., Chikka, M.R., Bhadra, U., Birchler, 914 J.A., Elgin, S.C. 2004. Heterochromatic silencing and HP1 localization in Drosophila are 915 dependent on the RNAi machinery. Science. 303(5658):669-72. 916 58. Pease, B., Borges, A.C., Bender, W. 2013. Noncoding RNAs of the Ultrabithorax domain 917 of the Drosophila bithorax complex. Genetics. 195(4):1253-64. 918 59. Petruk, S., Sedkov, Y., Riley, K.M., Hodgson, J., Schweisguth, F., Hirose, S., Jaynes, 919 J.B., Brock, H.W., Mazo, A. 2006. Transcription of bxd noncoding RNAs promoted by 920 trithorax represses Ubx in cis by transcriptional interference. Cell. 127(6):1209-21. 921 60. Pettini, T., Ronshaugen, M.R. 2016. Transvection and pairing of a Drosophila Hox long 922 noncoding RNA in the regulation of Sex combs reduced. bioRxiv. 045617. 923 61. Ponting, C.P., Belgard, T.G. 2010. Transcribed dark matter: meaning or myth? Human 924 Molecular Genetics. 19(R2):R162-8. 925 62. Ponting, C.P., Oliver, P.L., Reik, W. 2009. Evolution and functions of long noncoding 926 RNAs. Cell. 136: 629-41. 927 63. Pradeepa, M.M., McKenna, F., Taylor, G.C.A., Bengani, H., Grimes, G.R., Wood, A.J., 928 et al. 2017. Psip1/p52 regulates posterior Hoxa genes through activation of lncRNA 929 Hottip. PLoS Genet. 13(4). 930 64. Orii, H., Kato, K., Umesono, Y., Sakurai, T., Agata, K., Watanabe, K. 1999. The 931 planarian HOM/HOX homeobox genes (Plox) expressed along the anteroposterior axis. 932 Dev Biol. 210(2):456-68. 933 65. Qian, B., Wang, D.M., Gu, X.S., Zhou, K., Wu, J., Zhang, C.Y., He, X.Y. 2018. 934 LncRNA H19 serves as a ceRNA and participates in non-small cell lung cancer 935 development by regulating microRNA-107. Eur. Rev. Med. Pharmacol. Sci. 22:5946– 936 5953. 937 66. Rinn, J.L., Kertesz, M., Wang, J.K., Squazzo, S.L., Xu, X., Brugmann, S.A., Goodnough, 938 L.H., Helms, J.A., Farnham, P.J., Segal, E., Chang, H.Y. 2007. Functional demarcation 939 of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell. 940 129: P.1311-23. 941 67. Schmitz, S.U., Grote, P., Herrmann, B.J. 2016. Mechanisms of long noncoding RNA 942 function in development and disease. Cell. Mol. Life Sci. 73:2491–2509. 943 68. Shuaib, M., Parsi, K.M., Thimma, M., Adroub, S.A., Kawaji, H., Seridi, L., Ghosheh, Y., 944 Fort, A., Fallatah, B., Ravasi, T., Carninci, P., Orlando, V. 2019. Nuclear AGO1 945 Regulates Gene Expression by Affecting Chromatin Architecture in Human Cells. Cell 946 Syst. 9(5):446-458.e6. 947 69. Taft, R.J, Pheasant, M, Mattick, J.S. 2007. The relationship between non-protein-coding 948 DNA and eukaryotic complexity. Bioessays. 29:288–99. 949 70. Tang, W., Dong, K., Li, K., Dong, R., Zheng, S. 2016. MEG3, HCN3 and linc01105 950 influence the proliferation and apoptosis of neuroblastoma cells via the HIF-1 and p53 951 pathways. Sci. Rep. 6:36268. 952 71. Wang, K.C., Chang, H.Y. 2011. Molecular mechanisms of long noncoding RNAs. Mol 953 Cell. 43(6):904-14. 954 72. Wang, K.C., Yang, Y.W., Liu, B., Sanyal, A., Corces-Zimmerman, R., Chen, Y., Lajoie, 955 B.R., Protacio, A., Flynn, R.A., Gupta, R.A., Wysocka, J, Lei, M., Dekker, J., Helms, 956 J.A., Chang, H.Y. 2011. A long noncoding RNA maintains active chromatin to 957 coordinate homeotic gene expression. Nature. 472:120-4. 958 73. Wang, X., Lee, C., Gilmour, D.S., Gergen, J.P. 2007. Transcription elongation controls 959 cell fate specification in the Drosophila embryo. Genes Dev. 21:1031–1036. 960 74. Weinberg, M.S., Villeneuve, L.M., Ehsani, A., Amarzguioui, M., Aagaard, L., Chen, 961 Z.X., Riggs, A.D., Rossi, J.J., Morris, K.V. 2006. The antisense strand of small 962 interfering RNAs directs histone methylation and transcriptional gene silencing in human 963 cells. RNA. 12(2):256-62. 964 75. White, E., Schlackow, M., Kamieniarz-Gdula, K., Proudfoot, N.J., Gullerova, M. 2014. 965 Human nuclear Dicer restricts the deleterious accumulation of endogenous double- 966 stranded RNA. Nat Struct Mol Biol. 21(6):552-9. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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1003 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1004 Figures and Legends

1005 Figure 1. Schematic representation of Avi-Hox4 sequence with the projection of antisense cloned 1006 fragments of Avi-antiHox4_2 and Avi-antiHox4_1. Red color marks exons of the sense transcript. Blue 1007 color marks intergenic areas and introns, one of which is a part of Avi-antiHox4_1 exon. Light green color 1008 marks the exon of Avi-antiHox4_2, which is localized upstream to the sequence that codes Avi-Hox4 1009 mRNA. White color indicates an exon of the Avi-antiHox4_2 fragment, which was not found inside the 1010 known genomic sequence. The positions of introns and exons were obtained from genomic sequence 1011 locally assembled from short genomic reads using Geneious Prime software (data not shown).

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1024 Figure 2. Sense and antisense transcription of the Hox1 gene in juvenile A. virens. A. Schematic 1025 representation of the probes used with their projection onto the genomic sequence. B. Schematic 1026 representation of expression patterns for Avi-Hox1 (sense) and Avi-antiHox1 (antisense) transcripts 1027 (shown in green and orange respectively). C. WMISH with sense strand as probe. Expression of 1028 Avi-antiHox1 RNA in juvenile worm (a, b) and regenerating worm at 3 days after caudal amputation (c) 1029 (orange framework); White outline arrow in (a) points to the localized signal in the esophagus. D. (a-c) 1030 WMISH with antisense strand as probe. Expression of Avi-Hox1 mRNA in juvenile worm (a, b) and 1031 regenerating worm at 3 days after caudal amputation (c) (green framework). In C and D, the worms are 1032 oriented with their heads to the right on all photos. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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1050 Figure 3. Sense and antisense transcription of the Hox2 gene in juvenile A. virens. A. Schematic 1051 representation of the probes used with their projection onto the genomic sequence. B. Schematic 1052 representation of expression patterns for Avi-Hox2 (sense) and Avi-antiHox2 (antisense) transcripts 1053 (shown in green and orange respectively). C. WMISH with sense strand as probe detects Avi-antiHox2 1054 transcription in juvenile worms (a-c, orange framework). Back arrow (a) indicates dorsal semicircle of 1055 ectodermal cells with the signal; white arrow indicates the signal in esophagus; b - deep optical slice, 1056 demonstrating position of Avi-antiHox2 RNA in the ectodermal part of the growth zone; (c) presents 1057 expression in esophagus. D. WMISH with antisense strand as probe detects Avi-Hox2 transcription in 1058 juvenile worms (a-c, orange framework). (b) – deep optical slice, demonstrating position of Avi-Hox2 1059 mRNA in the mesodermal part of the growth zone; c presents the expression in the pharynx. The position 1060 of esophagus is marked by asterisk. In C and D, the worms are oriented with their heads to the right on all 1061 photos.

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1065 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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1082 Figure 4. Sense and antisense transcription of the Hox3 gene in P. dumerilii nectochaete. 1083 A. Schematic representation of the probes used with their projection onto the genomic sequence. 1084 B. WMISH with sense strand as probe detects Pdum-antiHox3 transcription in esophagus anlagen (orange 1085 framework, black outline arrow) (a); WMISH with antisense strand as probe detects Pdum-Hox3 1086 transcription in the forming growth zone (green framework) (b). B - head to the right.

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1097 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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1102 Figure 5. Sense and antisense transcription of the Hox4 gene at various larval stages of A. virens. 1103 A. Schematic representation of the probes used with their projection onto the genomic sequence. Purple 1104 color indicates the probe to the cloned sequence Avi-antiHox4_1. B. WMISH with sense strand as probe 1105 detects Avi-antiHox4 transcription at trochophore (a), metatrochophore (b) and nectochaete (c) stages 1106 (orange framework). C. WMISH with antisense strand as probe detects Avi-Hox4 transcription at the 1107 same stages as in B (green framework). D. WMISH with the probe to Avi-antiHox4_1 at trochophore 1108 (a-f), metatrochophore (g) and nectochaete (h) stages (purple framework). E. WMISH with antisense 1109 strand as probe detects Avi-Hox4 transcription at the same stages as in D (a-h; green framework). The full 1110 description of the transcription pattern is given in the text. B, C, D, E – ventral view; episphere to the top; 1111 (h) – head to the right.

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1118 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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1131 Figure 6. Sense and antisense transcription of the Hox4 gene in juvenile A. virens. A. WMISH with 1132 the probe to Avi-antiHox4_1 transcript (a, b; purple framework). WMISH with sense strand as probe 1133 detects 3’-associated Avi-antiHox4 transcript (c, d; orange framework); WMISH with antisense strand as 1134 probe detects Avi-Hox4 transcription (e, f; green framework); B. Schematic representation of expression 1135 patterns of all studied Hox4 transcripts in juvenile worms. A (a, c, e) – heads to the right. The full 1136 description is in the text.

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1147 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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1164 Figure 7. Sense and antisense transcription of the Hox4 gene at the various larval stages of 1165 P. dumerilii. A. Schematic representation of the probes used with their projection onto the genomic 1166 sequence. B. WMISH with sense strand as probe detects Pdum-antiHox4 transcript (a-c; orange 1167 framework). C. WMISH with antisense strand as probe detects Pdum-Hox4 transcript (a-с; green 1168 framework). (a) – ventral view, episphere to the top. (b, c) – ventral view, head to the right. The full 1169 description is in the text.

1170 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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1185 Figure 8. Sense and antisense transcription of the Hox5 gene at the larval stages A. virens. 1186 A. Schematic representation of the probes used with their projection onto the genomic sequence. Purple 1187 color indicates the probe to the cloned sequence of Avi-antiHox5. B. WMISH with the probe to 1188 Avi-antiHox5 transcript (a-p; purple framework). Early stage trochophore larvae (a, c, e, g) and middle 1189 stage trochophore larvae (i, k, m, o) are depicted in frontal projection (a, c, e, g, i, k, m, o) with the 1190 reducing depth of optical slices as the signal moves to the ventral side. In the bottom row the larvae are 1191 orientated with their vegetal pole to the front (b, d, f, h, j, l, n, p), ventral side is to the top. Red asterisk 1192 marks the vegetal pole. At C the enlarged fragment of B (d) is presented where the nuclear localization of 1193 the transcripts in two symmetrical domains is clearly visible. D. WMISH with sense strand as probe 1194 detects Avi-antiHox5 transcript (orange framework) at metatrochophore (a, b) and nectochaete (c-e) 1195 stages; WMISH with antisense strand as probe detects Avi-Hox5 transcript (green framework) at 1196 metatrochophore (f, g) and nectochaete (h-j) stages. On (d) and (i) the larvae of the same age are 1197 presented from the ventral side (left) and in lateral position (right, deep optical slice) to demonstrate the 1198 mesodermal localization of antisense transcript (white dotted line). Right square bracket marks the 1199 territory of the ventral neuroectoderm with no sense and antisense signal, the black asterisk marks the 1200 larval mouth. The full description of the transcription pattern is in the text.

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1202 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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1218 Figure 9. Co-localization of Avi-antiHox5 and Avi-Lox5 in A. virens trochophore. Sense and 1219 antisense transcription of the Hox5 gene in A. virens larvae. A. Double WMISH with the probe to Avi- 1220 antiHox5 and antisense strand as probe to Avi-Lox5 at the stage of middle trochophore (76 h). DIG- and 1221 FITC-probes to Avi-antiHox5 and Avi-Lox5 are detected with BM-Purple and FastRed, respectively. 1222 Vegetal view, ventral side to the top. B. WMISH with the probe to Avi-antiHox5 (purple framework) at 1223 the nectochaete stage. White outline arrows (b) indicate the unique Avi-antiHox5 expression at the basis 1224 of aciculae (supporting chaetae) (a, b). WMISH with sense strand as probe detects Avi-antiHox5 1225 transcript (orange framework) at the nectochaete stage (c, d); WMISH with antisense strand as probe 1226 detects Avi-Hox5 transcript (green framework) (e, f); on B (b), (d), (f) present deep optical slices. The full 1227 description of the transcription pattern is in the text.

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1233 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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1251 Figure 10. Sense and antisense transcription of the Hox5 gene in juvenile A. virens. A. WMISH with 1252 the probe to cloned Avi-antiHox5 (a-c, purple framework), sense strand as probe to Avi-Hox5 mRNA (d-f; 1253 orange framework) and antisense strand as probe to the same sequence (g-i, green frame). Head is to the 1254 right on all photos. Pygidium, growth zone and young segments are shown from the ventral side on b, e, 1255 h and on the deep optic slices on c, f, i. B. Schematic representation of expression patterns of all studied 1256 transcripts. The full description of the transcription pattern is in the text.

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1262 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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1274 Figure 11. Sense and antisense transcription of the Hox5 gene in juvenile P. dumerilii. A. Schematic 1275 representation of the probes’ position with the projection to genomic sequence. B. WMISH with sense 1276 strand as probe to reveal Pdum-Hox5 antisense transcript (orange framework). C. WMISH with antisense 1277 strand as probe to reveal Pdum-Hox5 mRNA (green framework). The full description of the transcription 1278 pattern is in the text.

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1290 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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1304 Figure 12. Sense and antisense transcription of the Hox7 gene in juvenile worms of A. virens. 1305 A. Schematic representation of the probes used with their projection onto the genomic sequence. The 1306 purple color indicates the fragment of the cloned Avi-antiHox7 (5’RACE), which probe does not reveal 1307 any transcription. B. Schematic expression patterns of studied transcripts of Avi-Hox7. C. WMISH with 1308 the sense strand as probe (a-d; orange frame). D. WMISH with the antisense strand as probe to reveal 1309 mRNA of Avi-Hox7 (a-b; green framework). On C (d) and D (d) the transcription pattern of antisense and 1310 sense RNA in the ventral ganglion of the formed segment (from the middle part of the worm) is shown. 1311 The white asterisk on C (c) marks the unspecific background in the intestine. The full description of the 1312 transcription pattern is in the text.

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1322 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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1337 Figure 13. Sense and antisense transcription of the Lox4 gene in juvenile P. dumerilii. A. Schematic 1338 representation of the probes used to 5’ and 3’- areas of mRNA with their projection onto the genomic 1339 sequence. B. Schematic representation of expression patterns of studied Pdum-Lox4 transcripts. 1340 C. WMISH with sense strand as probe to 3’-area of Pdum-antiLox4 transcript (a, b; orange framework). 1341 D. WMISH with antisense strand as probe to detect Pdum-Lox4 transcript (a, b; green framework). 1342 The full description of the transcription pattern is in the text.

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1347 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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1350 Figure 14. Sense and antisense transcription of the Lox2 gene in juvenile P. dumerilii. A. Schematic 1351 representation of the probes used with their projection onto the genomic sequence. B. Schematic 1352 representation of expression patterns of studied Pdum-Lox2 transcripts. C. WMISH with the sense strand 1353 as probe to reveal Pdum-antiLox2 transcription (a - d; orange frame) D. WMISH with the antisense strand 1354 as probe to reveal Pdum-Lox2 transcription (a, b; green framework). On C (c) and (d) the expression of 1355 Pdum-antiLox2 in the ganglion of the regenerating worm (10 hours post amputation) is shown. The full 1356 description of the transcription pattern is in the text.

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1363 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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1376 Figure 15. Sense and antisense transcription of the Post2 gene in A. virens larvae. A. Schematic 1377 representation of the probes used with their projection onto the genomic sequence. B. WMISH with the 1378 sense strand as probe to detect Avi-antiPost2 transcription (a-f, orange framework). C (a-b). WMISH 1379 with the sense strand as probe to detect Avi-antiPost2 transcription (orange framework). C (c-e). WMISH 1380 with the antisense strand as probe to detect Avi-Post2 transcription (green framework). All larvae have 1381 frontal orientation. C (a) presents the 90 hpf larva, view from the vegetal pole. C (b) (dotted frame) 1382 demonstrates the enlarged fragment of C (a) with the signal in two dots presumably in the nuclear. The 1383 full description of the transcription pattern is in the text.

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1394 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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1409 Figure 16. Sense and antisense transcription of the Post2 gene in larvae and juvenile worms of 1410 P. dumerilii. A. Schematic representation of the probes used with their projection onto the genomic 1411 sequence. B. WMISH with the sense strand as probe to detect Pdum-antiPost2 (a-d; orange frame). 1412 C. WMISH with the antisense strand as probe to detect Pdum-Post2 (a-d; green frame). On B (a) and C 1413 (a) the metatrochophores from the vegetal pole are shown. Dotted frames on B (c) and (d) and C (c) and 1414 (d) indicate the antisense and sense transcription in the regenerating worm (2 days post amputation), 1415 respectively. The full description of the transcription pattern is in the text.

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1425 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.30.428931; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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1427 Figure 17. Schematic representation of presumptive Pdum and Avi Hox cluster with the projections 1428 of probes' positions. Orange color indicates sense probes to mRNAs of corresponding Hox genes. 1429 Lavender color indicates the probes to the cloned NATs of Hox genes. The scheme contains the probes 1430 that detected expression patterns and two probes (marked by question mark) that gave controversial 1431 results for which more analysis is regarded. Blue outline marks the sequences of Hox genes. Shading: 1432 white – 5’ and 3’-UTRs; blue – protein coding areas; light blue – introns; shaded boxes – homeobox 1433 areas.

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