At the earliest stages of animal development, embryos consisting of only a handful of cells must figure out where each part of their body will come from. The first step in this process is to determine what will be their head versus their tail, and what will be their front versus their back. Many animals use specialized groups of cells, called “organizers”, to make this decision. This occurs in backboned animals – including humans – and also in distantly related animals such as spiders.

In spiders, the developing embryo must form an organizer called the “cumulus” or the spiderling will not develop correctly. In order to form and maintain the cumulus, various genes must be turned on in a carefully controlled order in exactly the right cells. Pechmann et al. have now discovered the role of a previously unknown gene (called Pt-Ets4) that marks the spot where the cumulus forms. This gene is required for cumulus maintenance and it also helps to activate a number of other cumulus-specific genes. When this gene is disrupted, the spider embryo does not properly differentiate its front from its back.

The findings presented by Pechmann et al. add to a growing foundation of studies aiming to understand how genes ‘talk’ to one another and organize embryos as they develop. In years to come, the unraveling of these gene pathways, where genes sequentially turn other genes on and off, will allow us to more fully understand how a single cell can grow into a complete adult animal.

The self-regulatory capacities of vertebrate embryos were most famously demonstrated by Spemann and Mangold. They found that by grafting the dorsal-lip of an amphibian embryo (now known as the Spemann Organizer) to the ventral side of the host gastrula embryo it was possible to induce a secondary body axis (Spemann and Mangold, 2001; De Robertis, 2009; Anderson and Stern, 2016).

Intriguingly, spider embryos also have high self-regulatory capacities, even to the extent that twinning can occur spontaneously (Napiórkowska et al., 2016; Oda and Akiyama-Oda, 2008). During spider embryogenesis a group of migratory cells (the cumulus) is needed to break the radial symmetry of the early embryo and to induce the dorsoventral body axis (Oda and Akiyama-Oda, 2008; Akiyama-Oda and Oda, 2003, 2006; McGregor et al., 2008; Hilbrant et al., 2012; Mittmann and Wolff, 2012; Schwager et al., 2015). Similar to the vertebrate experiments, Holm showed that transplanting cumulus material was able to induce a secondary axis in spider embryos (Holm, 1952). Modern work has shown that the cumulus signals via BMP signaling (again, similar to vertebrates). The mesenchymal cumulus cells are the source of the BMP receptor ligand Decapentaplegic (Akiyama-Oda and Oda, 2006). Interfering with the BMP signaling pathway by gene knockdown results in the loss of dorsal tissue identity, which in turn leads to completely radially-symmetric and ventralized embryos (Akiyama-Oda and Oda, 2006). The cumulus forms in the center of the so-called germ-disc (the embryonic pole of the embryo) and migrates underneath the ectoderm towards the rim of the disc. Arrival of the cumulus at the rim induces the opening of the germ-disc (Oda and Akiyama-Oda, 2008; Akiyama-Oda and Oda, 2003, 2006; McGregor et al., 2008; Hilbrant et al., 2012; Mittmann and Wolff, 2012; Schwager et al., 2015). Cumulus migration is dependent on the Hh-signaling pathway (Akiyama-Oda and Oda, 2010) and it was shown that the knockdown of components of this signaling pathway results in cumulus migration defects and in the ectopic opening of the germ-disc (Akiyama-Oda and Oda, 2010).

How the cumulus is specified and forms is still under debate. During the formation of the germ-disc a small cluster of cells ingress and form an indentation where the future center of the fully formed germ-disc will be located. This cluster of cells appears as a visible spot and is called the primary thickening (Akiyama-Oda and Oda, 2003; Hilbrant et al., 2012). However, it is not clear whether all or only a subset of the cells of the primary thickening give rise to the cumulus, or if cumulus cells arise from subsequent cell invagination at the site of the primary thickening (Oda and Akiyama-Oda, 2008; Akiyama-Oda and Oda, 2003). Cell tracing (Holm, 1952; Edgar et al., 2015), as well as the expression of the endodermal marker forkhead (Oda et al., 2007) within the primary thickening/cumulus cells led to the suggestion that the primary thickening/cumulus cells are central endodermal cells (Hilbrant et al., 2012; Oda et al., 2007). However, these studies could not completely rule out that the labeled cumulus cells develop into cells of the visceral mesoderm (Edgar et al., 2015).

During the last 15 years, research focused on candidate genes known to be involved in development in Drosophila melanogaster has revealed several aspects of how spider embryos pattern their main body axis. However, there are many open questions regarding the early regulation of cumulus specific gene expression, cumulus establishment and maintenance.

To overcome the limitations of the candidate gene approach, we have carried out transcriptome sequencing of carefully staged embryos to find new genes involved in cumulus and axial patterning in the spider Parasteatoda tepidariorum. From this work, we have identified the transcription factor Pt-Ets4 as a new gene expressed during early development and have found it to be expressed exclusively within the central primary thickening and the cells of the migrating cumulus. Our combined genetic and cellular analyses show that Pt-Ets4 is needed for the integrity of the cumulus. We found that the knockdown of this gene leads to embryos that show axis patterning defects reminiscent of BMP knockdown phenotypes, suggesting that an intact cumulus is needed to induce the formation of the bilaterally symmetric spider embryo. Importantly, Pt-Ets4 is necessary and sufficient for driving the early expression of twist (a gene involved in gastrulation and mesoderm formation in Drosophila) and hunchback, and the ectopic expression of Pt-Ets4 is sufficient to induce cell delamination.

The formation of the germ-disc is one of the most important events during spider embryogenesis. While a regular blastoderm (with no visible axial polarity) is present at stage 2, the germ-disc condenses during stage 3 of embryonic development (Figure 1A and B, for detailed description see [Pechmann, 2016]). This event leads to the establishment of the anterior/posterior body axis (anterior: rim of the disc; posterior: center of the disc).

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To find new genes that are involved in the process of axis specification we sequenced the embryonic transcriptomes of stage 1, stage 2 and stage 3 embryos (Figure 1—figure supplement 1) and searched for genes showing a similar expression profile as Pt-decapentaplegic (Pt-dpp), Pt-hedgehog (Pt-hh) and Pt-patched (Pt-ptc) (representing genes that show early transcription and are required for axis formation or cumulus migration, respectively (Akiyama-Oda and Oda, 2006, Akiyama-Oda and Oda, 2010); Figure 1—figure supplement 1). One of the candidates was an Ets-like gene with high similarity to Drosophila melanogaster Ets4/Ets98B (see Figure 1—figure supplement 2), henceforth called Parasteatoda tepidariorum Ets4 (Pt-Ets4).

Pt-Ets4 is necessary for the integrity of the cumulus

Time-lapse imaging and cross-sectioning revealed that the knockdown of Pt-Ets4 neither affected formation of the germ-disc nor of the primary thickening/cumulus (Video 1B; middle column in Figure 2, Figure 3A and B). However, during stage 5, cumulus integrity was affected in Pt-Ets4 knockdown embryos (Video 1B, middle column in Figure 2). While in control embryos the cumulus migrated towards the rim (Video 1A, left column in Figure 2), the cumulus of Pt-Ets4 RNAi embryos remained at the center of the germ-disc until early stage 5 and disappeared soon after gastrulation was initiated at the center and at the rim of the disc (Video 1B (15h onwards), middle column in Figure 2). Analysis of mid-stage 5 Pt-Ets4 RNAi embryos for the expression of the cumulus marker Pt-fascin (Akiyama-Oda and Oda, 2010) revealed that although the cells of the cumulus were still in the center of the germ-disc, they appeared to be more loosely organized (Figure 3C and D).

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Video 1

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In Pt-Ets4 knockdown embryos the radial symmetry of the germ-disc was not broken (as shown by the formation of a tube-like germ band in Video 1B (30h onwards), and the middle column in Figure 2), presumably due to the loss of the cumulus. To investigate this phenotype in more detail, we knocked down another gene that also results in defects in radial symmetry breaking, Pt-ptc. In Pt-ptc knockdown embryos, cumulus migration is lost but the cumulus itself is otherwise unaffected (Akiyama-Oda and Oda, 2010). After pRNAi with Pt-ptc, the cumulus stays in the center of the germ-disc (Video 1C; right column in Figure 2) and BMP signaling is ectopically activated (as shown via antibody staining against the phosphorylated form of mothers against dpp (pMAD) (Akiyama-Oda and Oda, 2010)). As a result, the germ-disc ectopically opened at the center and the dorsal field was induced at the posterior pole of the Pt-ptc RNAi embryos (right column in Figure 2). This ectopic induction of the dorsal field in the center of the germ-disc was never observed in the Pt-Ets4 RNAi embryos. To test if the disappearing cumulus was at least partially able to activate BMP signaling in the germ-disc of Pt-Ets4 RNAi embryos, we performed a pMAD antibody staining in both control and Pt-Ets4 RNAi embryos. In control embryos the cumulus reached the periphery of the germ-disc at late stage 5 and a strong pMAD staining was visible in the overlying ectodermal cells (Figure 3E, Figure 3—figure supplement 1). At this stage, the anterior marker Pt-orthodenticle (Pt-otd) was expressed in a ring, which had a width of 3–5 cells (Akiyama-Oda and Oda, 2003; Pechmann et al., 2009; Akiyama-Oda and Oda, 2016) (Figure 3E). In Pt-Ets4 knockdown embryos Pt-otd expression was unaffected but nuclear pMAD was not detectable (Figure 3F). This lack of BMP signaling explains why Pt-Ets4 RNAi embryos do not induce the dorsal field and stay radially symmetric. Indeed, the knockdown embryos were completely ventralized; the expression of the ventral marker Pt-short-gastrulation (Pt-sog) was uniform around the embryonic circumference and the segmental marker Pt-engrailed (Pt-en) was expressed in symmetric rings demonstrating the radial symmetry of the embryo (Figure 3G–L). During later development the embryonic tissue either grew completely over the yolk (Figure 3J–L), or a tube like structure elongated at the posterior of the embryo (Video 1B, middle column Figure 2). This was in contrast to Pt-ptc RNAi embryos, where the germ-disc opened up centrally and a tube like structure formed at the anterior of the embryo (right column Figure 2; Video 1C).

The Pt-Ets4 knockdown phenotype is rather similar to that of Pt-dpp, although cumulus migration/integrity is not affected in Pt-dpp RNAi embryos (Akiyama-Oda and Oda, 2006). This observation, plus the early and strong expression of Pt-Ets4 and the fact that BMP signaling disappeared upon Pt-Ets4 knockdown, led us to hypothesize that Pt-Ets4 functions as an activator of Pt-dpp transcription within the cells of the cumulus. However, there was no obvious difference in the expression of Pt-dpp in stage 4 control and Pt-Ets4 RNAi embryos (Figure 4A and E). Vice versa, Pt-dpp appears not to be regulating the expression of Pt-Ets4 (Figure 4—figure supplement 1A). In addition, Hh signaling seems not to be involved in the regulation of Pt-Ets4 expression and Pt-Ets4 appears not to regulate the expression of Pt-ptc within the primary thickening (Figure 4—figure supplement 1B and C).

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In order to determine what other genes Pt-Ets4 may regulate, we studied the expression of genes normally expressed in the primary thickening in Pt-Ets4 RNAi embryos. We found that Pt-forkhead (Pt-fkh) expression was unaffected (Figure 4B and F), while the cumulus marker Pt-fascin was slightly down regulated, and Pt-hunchback (Pt-hb) was strongly down regulated in Pt-Ets4 knockdown embryos (Figure 4C,D,G and H).

Ectopic expression of Pt-Ets4 induces cell migration

As Pt-Ets4 is strongly expressed in the cumulus and is required for cumulus integrity, we wondered how ectopic expression of Pt-Ets4 would affect cell behavior. To generate small cell clones ectopically expressing Pt-Ets4 within the germ-disc, we micro-injected late stage 1 embryos (via single cell/blastomere injections [Hilbrant et al., 2012; Kanayama et al., 2010]) with in vitro synthesized capped mRNA coding for an EGFP-Pt-Ets4 fusion protein (see Figure 5—figure supplement 1).

Our first observation was that the EGFP-Pt-Ets4 fusion protein localizes to the nuclei of the injected cells, suggesting that the nuclear-localization-signal of Pt-Ets4 is functioning normally (Figure 5A). EGFP-Pt-Ets4 marked cell clones resembled wild-type until stage 4. As soon as a dense germ-disc formed, however, the cell clones expressing EGFP-Pt-Ets4 seemed to move beneath the germ-disc and the EGFP signal became occluded by the opaque cells of the germ-disc (Figure 5B). We have never seen such behavior following injection of EGFP-NLS constructs alone (Figure 5F–F’’’), indicating that the change in cell behavior is due to the ectopic expression of Pt-Ets4.

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To further visualize the process of cell clone delamination, we marked cell membranes by co-injecting capped mRNA coding for EGFP-Pt-Ets4 together with capped mRNA coding for lynGFP (Köster and Fraser, 2001). In control embryos (ectopic expression of lynGFP alone), lynGFP strongly marked the cell outlines and cell clones stayed at the surface epithelium of the germ-disc (Video 2A, Figure 5C–C’’). Regardless of the position and the shape of the cell clone, cells expressing lynGFP/EGFP-Pt-Ets4 constricted and delaminated shortly after the formation of the germ-disc (Video 2B–D, Figure 5D–D’’). The detection of EGFP-Pt-Ets4 cell clones in fixed embryos using an antibody against EGFP confirmed that the labeled cells were below the epithelium of the germ-disc (Figure 5E and E’).

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The nuclear signal of the EGFP-Pt-Ets4 fusion construct (and the membrane signal of the lynGFP) is hardly visible after the delamination process. Therefore, we co-injected capped mRNA for EGFP-Pt-Ets4 with capped mRNA for nuclear localized EGFP (EGFP-NLS, see Figure 5—figure supplement 1), a construct that we have found to produce a very bright and persistent fluorescent signal. This experiment resulted in a strong nuclear localized EGFP signal within the marked cell clone, which we used to perform time-lapse imaging. While in the control embryos (injected with EGFP-NLS alone) the marked cell clones persisted at the surface of the germ-disc (Video 3A, Figure 5F and F’), the cell clone expressing both EGFP-NLS and EGFP-Pt-Ets4 delaminated shortly after the formation of the germ-disc (Video 3B, Figure 5G and G’). As previously shown (Kanayama et al., 2010, Kanayama et al., 2011), germ-disc cells continue to divide and undergo convergent extension during the formation of the germ-band, which causes cell clones to become thin and elongated as seen in our control EGFP-NLS clones (Video 3A, Figure 5F’’ and F’’’). In contrast to this, cell clones expressing both EGFP-NLS and EGFP-Pt-Ets4 stopped dividing as soon they delaminated. In addition, when the cumulus started to migrate, the delaminated cells of the EGFP-NLS/EGFP-Pt-Ets4 marked cell clone lost contact with each other and spread out underneath the germ-disc/germ-band epithelium (Video 3B, Figure 5G’’ and G’’’). This observation was consistent and reproducible in multiple analyzed embryos (Video 4).

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These results demonstrate that Ets4 expression is sufficient to induce cell delamination and migration in embryos of P. tepidariorum.

Ectopic expression of Pt-Ets4 induces mesoderm-like fate

As already mentioned, Pt-Ets4 seems to have no influence on the early expression of Pt-dpp itself (Figure 4A and E). Furthermore, we were not able to detect Pt-dpp transcripts in cells ectopically expressing Pt-Ets4 (Figure 6A and A’). In contrast to Pt-dpp, we did find that Pt-Ets4 is regulating the expression of Pt-hb. While Pt-hb expression was nearly absent in Pt-Ets4 RNAi embryos (Figure 4H), Pt-hb transcripts were present in the ectopically Pt-Ets4 positive cell clone (Figure 6B and B’).

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The behavior of the Pt-Ets4 positive cells is reminiscent of migrating gastrulating cells that invade the germ-disc from the center and from the rim of the germ-disc (Mittmann and Wolff, 2012; Kanayama et al., 2011; Yamazaki et al., 2005) (see Video 1A). As Pt-Ets4 activates the expression of Pt-hb (a gene that is known to be expressed in mesodermal cells in diverse animals [Schwager et al., 2009; Franke and Mayer, 2015; Kerner et al., 2006]) we wondered if Pt-Ets4 misexpression is inducing a mesoderm-like cell fate. For this reason, we tested whether the ectopic expression of Pt-Ets4 is also inducing the expression of the key mesodermal marker Pt-twist (Pt-twi). Indeed, Pt-twi was detectable within the cell clone that ectopically expressed Pt-Ets4 (Figure 6C and C’, compare to controls in Figure 6—figure supplement 1). Interestingly, in the stage 4 embryos ectopically expressing Pt-Ets4, we could not only detect Pt-twi transcripts within the ectopic Pt-Ets4 expressing cell clone, but also within the central primary thickening (Figure 6C’). This comes as a surprise, as it was reported that Pt-twi expression is not initiated before the end of stage 5 (Yamazaki et al., 2005). For this reason, we reanalyzed the full expression series of Pt-twi in wild-type embryos and we were able to confirm that Pt-twi is expressed in the developing primary thickening of stage 3 and 4 embryos (Figure 6—figure supplement 2A–C). Finally, we confirmed the regulation of Pt-twi via Pt-Ets4 by analyzing the expression of Pt-twi in Pt-Ets4 knockdown embryos. Pt-twi transcripts were no longer detectable in the primary thickening of stage 4 Pt-Ets4 RNAi embryos (Figure 6D and E). However, late segmental mesoderm specification was unaffected, as the expression of Pt-twi was unchanged in stage 7 Pt-Ets4 knockdown embryos (Figure 6—figure supplement 2H–I’). This confirms that the formation and migration of the cumulus and later gastrulation events from central and peripheral parts of the germ-disc are two independent processes (Hilbrant et al., 2012; Oda et al., 2007).

Overall, we suggest that the activation of Pt-twi and Pt-hb by Pt-Ets4 in cell clones initiated a mesoderm-like cell fate that led to the migratory behavior of the ectopic Pt-Ets4 positive cells.

Ets proteins belong to a highly conserved family of transcription factors (Laudet et al., 1999) that play roles in a variety of different cellular processes (e.g. growth, migration, differentiation) and it has been shown that these factors can act as activators or repressors of transcription (Oikawa and Yamada, 2003; Sharrocks et al., 1997; Sharrocks, 2001; Hollenhorst et al., 2011). In D. melanogaster, Ets4 (also known as Ets98B) is expressed in the oocyte nucleus and the primordial germ cells (PGC) (Chen et al., 1992; Hsouna et al., 2004) and there is evidence that Dm-Ets4 is involved in the migration of the PGCs (Hsouna et al., 2003). Furthermore, the mammalian homolog of Ets4, Pdef, is down regulated in invasive and migratory breast tumor cells (Feldman et al., 2003). Together, these reports suggest that Ets4 regulates migratory cell behavior in different organisms.

Here we show that in the spider P. tepidariorum, Ets4 is strongly expressed in the migrating cumulus and that Pt-Ets4 is needed for the integrity of the cumulus, a group of cells, which need to migrate together in order to function normally. We also show that the ectopic expression of Pt-Ets4 is able to induce cell delamination and cell migration within the ectodermal germ-disc cells (a process that is known as epithelial-to-mesenchymal transition [Thiery et al., 2009; Gilmour et al., 2017]). Taken together, these findings suggest that Pt-Ets4 likely plays an important role in the migratory behavior of the cumulus.

It has been suggested that the cumulus cells of many higher spiders are specified during early embryogenesis and are not secondarily induced after the formation of the germ-disc (Edgar et al., 2015). Pt-Ets4 marks the cells of the developing primary thickening (st. 3 and 4) and of the migrating cumulus. Therefore, our results indicate that in P. tepidariorum as well, cumulus cells are induced before germ-disc formation is complete.

As the primary thickening forms in the absence of Pt-Ets4, Pt-Ets4 cannot be the only factor that is required for the specification of the cells that will develop into the primary thickening (and later into the cumulus). This is supported by our finding that Pt-Ets4 is sufficient to induce the delamination and migration of ectodermal cells, but it is not sufficient to induce the formation of a fully functional ectopic cumulus. So far, there have been no reports of any gene knockdown that completely inhibits the formation of the primary thickening. The cumulus is characterized by the co-expression of multiple genes, including Pt-dpp. As shown by our analyses, Pt-Ets4 seems not to be involved in the regulation of Pt-dpp itself, as well as several other cumulus marker genes. Therefore, important factors of the cumulus are missing in the ectopic Pt-Ets4 expression cells. Taken together, our findings indicate that the formation of the primary thickening is a process that is buffered via the input of several genes or other unknown mechanisms/factors.

Our results also show that an intact cumulus is needed to open up the germ-disc and to initiate the formation of a bilaterally symmetric spider embryo. In the germ-disc of P. tepidariorum embryos, BMP signaling is active from early stage 5 (Figure 3—figure supplement 1). However, the germ-disc does not open up before the arrival of the cumulus at the rim of the disc at the end of stage 5 even though the putative receptors are ubiquitously expressed. Therefore, the establishment of the dorsal field is a precisely timed process, and it is possible that in Pt-Ets4 knockdown embryos the cells of the cumulus disperse too early, and are no longer able to induce the opening of the germ-disc at the end of stage 5. An alternative, although not mutually exclusive, explanation involves the fact that the cumulus may signal via cytonemes (Akiyama-Oda and Oda, 2003; Hilbrant et al., 2012). These structures could be affected in Pt-Ets4 RNAi embryos, as the cumulus cells lose contact with each other. Lastly, Pt-Ets4 could be involved in regulating the BMP pathway activity at a different level (e.g. protein-protein interactions, protein modifications) or the knockdown of Pt-Ets4 could result in a fate change of the cumulus cells before cumulus migration is initiated. Investigation of each of these hypotheses will require the establishment of advanced techniques to study cell migration, cell microstructure, and protein interactions in P. tepidariorum.

Via both our loss- and gain-of-function experiments, we found that Pt-Ets4 is involved in the activation of at least two genes that are expressed within the primary thickening of stage 4 embryos. These genes are Pt-hb and Pt-twi. For Pt-hb, it was already shown that this gene is strongly expressed within the primary thickening of stage 4 embryos (Schwager et al., 2009). However, it was reported that Pt-twi expression is not detectable before late stage 5 (Yamazaki et al., 2005), while we found that Pt-twi is expressed in the primary thickening already at stage 3 and 4. This is in agreement with our sequencing data, which shows that Pt-twi is strongly up regulated from stage 2 to stage 3 (Figure 1—figure supplement 1). This result indicates that the cumulus cells are not endodermal in nature (Oda and Akiyama-Oda, 2008; Hilbrant et al., 2012; Oda et al., 2007) but rather are mesendodermal. For this reason, the genetic composition and the eventual fate of the cumulus should be closely examined in future studies.

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Author details

1. Matthias Pechmann

   Developmental Biology, Institute of Zoology, University of Cologne, Cologne, Germany

   Contribution

   MP, Conceived the project, designed and performed the experiments, interpreted the results and wrote the initial draft of the paper

   For correspondence

   pechmanm@uni-koeln.de

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-0043-906X

2. Matthew A Benton

   Developmental Biology, Institute of Zoology, University of Cologne, Cologne, Germany

   Contribution

   MAB, Designed and performed the experiments. Performed the phylogenetic analysis. Interpreted the results, contributed to the writing of the final manuscript and approved the final manuscript

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-7953-0765

3. Nathan J Kenny

   Life Sciences Department, The Natural History Museum, London, United Kingdom

   Contribution

   NJK, Performed the bioinformatic analyses. Contributed to the writing of the final manuscript and approved the final manuscript

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-4816-4103

4. Nico Posnien

   Department of Developmental Biology, University of Goettingen, Goettingen, Germany

   Contribution

   NP, Performed the bioinformatic analyses. Contributed to the writing of the final manuscript and approved the final manuscript

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-0700-5595

5. Siegfried Roth

   Developmental Biology, Institute of Zoology, University of Cologne, Cologne, Germany

   Contribution

   SR, Designed the experiments. Interpreted the results. Contributed to the writing of the final manuscript and approved the final manuscript

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-5772-3558

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