One of the most fundamental sensory cell types that emerged in animal evolution is the mechanosensory cell – the specialized sensory epithelial cell that transduces mechanical stimuli (e.g. water vibration) into internal signals. These signals are then communicated, usually via the nervous system, to effector cells (e.g. muscle cells) to elicit behavioral and/or physiological responses of the organism. Indeed, specialized mechanosensory cells are found across diverse animal lineages, from vertebrate hair cells, cephalopod angular acceleration receptors, to statocyst cells of cnidarian jellyfish and ctenophores. Typically, a mechanosensory cell bears an apical mechanosensory apparatus consisting of a single non-motile cilium surrounded by a circle of rigid microvilli with actin rootlets (i.e. stereovilli, or stereocilia), and extends basal neuronal processes that connect to the nervous system (reviewed in Beisel et al., 2008; Budelmann, 1989; Manley and Ladher, 2008).

The structure of animal mechanosensory cells is not uniform, however (reviewed in Bezares-Calderón et al., 2020). For instance, insect and cephalopod mechanosensory cells lack stereovilli (Jarman, 2002; Budelmann, 1989), while the apical mechanosensory apparatus of vertebrate hair cells is differently shaped, having a cilium on one side of a group of stereovilli of graded lengths, with the stereovilli next to the cilium being the longest (Fain, 2003). The observed morphological diversity in mechanosensory cells of distantly related animals has led to a fundamental question in animal mechanoreceptor evolution: whether the diversity evolved by divergence from a common ancestral form (Beisel et al., 2008; Jørgensen, 1989; Schlosser, 2020), or by independent evolution (Coffin et al., 2004; Holland, 2005). Addressing this question requires an understanding of the mechanisms of mechanoreceptor development across disparate groups of animals.

Developmental genetics of mechanosensory cells has been extensively studied in bilaterian models such as vertebrates and flies (reviewed in Schlosser, 2020; Boekhoff-Falk, 2005; Beisel et al., 2008). Yet, relatively little is known about the genetics of mechanoreceptor development in non-bilaterian, early-evolving animal groups such as Cnidaria (e.g. jellyfish, corals, and sea anemones), Ctenophora (combjellies), Placozoa and Porifera (sponges), the knowledge of which is key to defining the ancestral conditions for mechanoreceptor development basal to Bilateria. This baseline knowledge, in turn, is necessary for reconstructing how mechanoreceptors diversified in each lineage. In this paper, we focus on investigating the development of a fundamental, yet understudied, mechanosensory cell type of Cnidaria – the concentric hair cell.

Cnidaria is the sister group to Bilateria (Medina et al., 2001; Putnam et al., 2007; Hejnol et al., 2009; Erwin et al., 2011), and has two broad classes of mechanosensory cells – cnidocytes (Brinkmann et al., 1996) and concentric hair cells (Arkett et al., 1988; Oliver and Thurm, 1996; Holtmann and Thurm, 2001; not to be confused with vertebrate hair cells) – that are characterized by an apical mechanosensory apparatus consisting of a single cilium surrounded by a ring of stereovilli. The cnidocyte is the phylum-defining stinging cell type, and additionally contains a cnidarian-specific exocytotic organelle called the cnida (plural: cnidae) which is made up of a capsule enclosing a coiled tubule (reviewed in Thomas and Edwards, 1991; Lesh-Laurie and Suchy, 1991; Fautin and Mariscal, 1991). Cnidocytes are abundant in the ectodermal epithelium of cnidarian tentacles, and, upon perceiving mechanical stimuli, discharge cnidae by rapidly everting the coiled tubule to pierce nearby animals for defense and/or prey capture. There is no structural or functional evidence that the cnidocyte transmits sensory information to other cells, but firing of cnidae is thought to be modulated by neurons that innervate cnidocytes through chemical synapses (Westfall, 2004). Thus, the cnidocyte is a cnidarian-specific mechanosensory cell type that – uniquely among animal mechanosensory cells – functions as an effector cell.

The cnidarian hair cell, on the other hand, represents the classical mechanosensory cell type with dedicated sensory-neuronal function. Hair cells are integrated within the ectodermal epithelium of mechanosensory structures, such as gravity sensors of jellyfishes and tentacles of hydroids and corals (Horridge, 1969; Lyons, 1973; Tardent and Schmid, 1972; Singla, 1975; Hündgen and Biela, 1982). Structurally, the cnidarian hair cell exhibits the stereotypical mechanosensory neuron-like morphology described above, including the apical mechanosensory apparatus and basal neurites that become part of the basiepithelial nerve plexus (Horridge, 1969; Singla, 1975; Singla, 1983; Hündgen and Biela, 1982). Upon stimulation, the hair cells communicate mechanosensory information to other cells by converting mechanical stimuli into internal electrical signals (Arkett et al., 1988; Oliver and Thurm, 1996), and are thought to generate highly coordinated response behaviors such as righting and feeding. Similar to vertebrate hair cells, hair cells of jellyfish gravity sensors are sensitive to sound and can be lost due to noise trauma (Solé et al., 2016). Cnidarian hair cells show morphological and functional characteristics that parallel those of mechanosensory cells in other animal lineages, consistent with a deep evolutionary origin or convergent origins.

Although genetics of cnidocyte development is increasingly understood (e.g. Babonis and Martindale, 2017; Richards and Rentzsch, 2015; Richards and Rentzsch, 2014; Wolenski et al., 2013), that of cnidarian hair cell development remains poorly known. This knowledge gap severely limits our ability to reconstruct the evolutionary histories of mechanoreceptor development within Cnidaria and across the basal branches of the animal tree. A previous study has shown that the class IV POU homeodomain transcription factor (POU-IV or Brn-3)-encoding gene is expressed in the hair-cell-bearing mechanosensory organ called the touch plate in moon jellyfish Aurelia sp.1 (Nakanishi et al., 2010), consistent with a role in cnidarian hair cell development. Yet, the function of POU-IV in cnidarian hair cell development, if any, remains undefined. As the first step toward elucidating the genetic mechanism of cnidarian hair cell development, here we dissect the role of POU-IV in the development of mechanosensory hair cells using the genetically tractable sea anemone cnidarian model Nematostella vectensis.

POU-IV is shared by all extant animal groups except for Ctenophora (comb jellies), indicative of early emergence in animal evolution (Gold et al., 2014). POU-IV is absent in choanoflagellates, although the class II POU-like gene has been reported to be present in Mylnosiga fluctuans indicative of a premetazoan origin of POU transcription factors (López-Escardó et al., 2019). As in other POU proteins, POU-IV is characterized by having a bipartite DNA-binding domain consisting of the N-terminal POU-specific domain and the C-terminal POU homeodomain (reviewed in Herr and Cleary, 1995). In Bilateria, POU-IV-binding DNA elements are POU-IV-class-specific and conserved; mammalian POU-IV proteins Brn3.0 (Brn-3a or POU4F1) and Brn3.2 (Brn-3b or POU4F2) and Caenorhabditis elegans POU-IV protein Unc-86 bind to a highly symmetrical core sequence AT(A/T)A(T/A)T(A/T)AT (Gruber et al., 1997). In bilaterian animal models such as C. elegans, POU-IV is known to function as a terminal selector – a transcription factor that determines mature cell identity via direct regulation of effector genes (reviewed in Leyva-Díaz et al., 2020). The cell type whose identity is defined by POU-IV across bilaterian lineages is the mechanosensory cell. In humans, mutations at one of the pou-iv loci – Brn-3c (Brn3.1 or POU4F3) – have been linked to autosomal dominant hearing loss (Vahava et al., 1998), and in Brn-3c knockout mice, auditory and vestibular hair cells fail to complete differentiation (Erkman et al., 1996; Xiang et al., 1997b) and are lost by cell death (Xiang et al., 1998). Likewise, in C. elegans, the pou-iv ortholog (unc-86) regulates differentiation of mechanosensory touch cells (Chalfie and Sulston, 1981; Chalfie and Au, 1989; Finney and Ruvkun, 1990; Duggan et al., 1998). In addition to its role in mechanoreceptor differentiation, POU-IV defines the identity of olfactory chemosensory neurons in Drosophila (Clyne et al., 1999), as well as retinal ganglion cells (Brn-3b; Erkman et al., 1996; Gan et al., 1996) and subsets of CNS neurons in mice (Brn-3a; Serrano-Saiz et al., 2018; McEvilly et al., 1996; Xiang et al., 1996). In Cnidaria, POU-IV is expressed not only in the developing mechanoreceptor of Aurelia sp.1 (Nakanishi et al., 2010) as described above, but also in the statocysts of the freshwater hydrozoan jellyfish Craspedacusta sowerbii (Hroudova et al., 2012). Also, POU-IV is required for postmitotic differentiation of cnidocytes, as well as elav::mOrange neurons, in N. vectensis (Tournière et al., 2020). Consistent with cnidarian POU-IV being a terminal selector, a genome-wide analysis of differential gene expression between POU-IV knockout mutant N. vectensis and their siblings indicates that POU-IV controls the expression of effector genes that define mature neural identity, such as those involved in ion channel activity (Tournière et al., 2020). However, it remains unknown if cnidarian POU-IV directly regulates effector gene expression, as expected for a terminal selector. Furthermore, although POU-IV recognition element-like sequences have been previously identified in the N. vectensis genome based on sequence similarity to bilaterian POU-IV-binding motifs (Sebé-Pedrós et al., 2018), cnidarian POU-IV recognition elements have not been experimentally defined, and consequently, whether the conservation of POU-IV-binding sequence extends beyond Bilateria remains unclear.

Sea anemones together with corals form the clade Anthozoa, which is sister to the Medusozoa – a group characterized by typically having a pelagic medusa (jellyfish) stage – consisting of Staurozoa, Hydrozoa, Scyphozoa, and Cubozoa (Collins et al., 2006; Zapata et al., 2015). Sea anemones have multicellular mechanosensory structures, known as the hair bundle mechanoreceptors, in the ectoderm of the oral feeding tentacles (Mire-Thibodeaux and Watson, 1994; Mire and Watson, 1997; Watson et al., 1997). A hair bundle mechanoreceptor consists of a central sensory cell surrounded by peripheral support cells (Figure 1—figure supplement 1). The central sensory cell exhibits morphological hallmarks of concentric hair cells, with an apical cilium surrounded by stereovilli, and basal neurites. Support cells contribute stereovilli or microvilli that encircle the apical ciliary-stereovillar structure of the central hair cell. The cilium and stereovilli of the central cell and stereovilli/microvilli of support cells are interconnected by lateral linkages; in addition, extracellular linkages have been observed between the tips of stereovilli/microvilli of support cells, resembling the tip links of vertebrate mechanosensory hair cells (Watson et al., 1997). The apical sensory apparatus, or the hair bundle, of the mechanoreceptor thus consists of the cilium and stereovilli of the central hair cell and the peripheral stereovilli/microvilli of support cells (Mire and Watson, 1997). We note that in the literature, the support cells of hair bundle mechanoreceptors are sometimes referred to as hair cells (e.g. Mire and Watson, 1997). In this paper, in accordance with the morphological definition of cnidarian hair cells, a hair cell refers to the central sensory cell of the hair bundle mechanoreceptor, and a support cell refers to the cell that abuts the central sensory cell and contributes peripheral stereovilli/microvilli to the hair bundle.

In this report, we use the starlet sea anemone N. vectensis to investigate the role of POU-IV in the development of cnidarian hair cells. N. vectensis is a convenient model for studies of mechanisms of cnidarian development because of the availability of the genome sequence (Putnam et al., 2007) and a wide range of powerful molecular genetic tools including CRISPR-Cas9 genome editing (Ikmi et al., 2014; Nakanishi and Martindale, 2018). During embryogenesis, N. vectensis gastrulates by invagination to form an embryo consisting of ectoderm and endoderm separated by the extracellular matrix known as the mesoglea (Kraus and Technau, 2006; Magie et al., 2007). The blastopore becomes the mouth/anus (‘oral’) opening of the animal (Fritzenwanker et al., 2007; Lee et al., 2007). The embryo develops into a free-swimming, ciliated planula larva, which transforms into a polyp with circumoral tentacles that house mechanosensory hair cells in the ectoderm (Figure 1—figure supplement 2; Nakanishi et al., 2012; Watson et al., 2009). The polyp then grows and reaches sexual maturity. Previous studies have indicated that hair bundles of N. vectensis polyps are indeed sensitive to movement of surrounding water (Watson et al., 2009), and that stereovilli/microvilli of hair bundles express TRP (transient receptor potential)-like cation channels (Mahoney et al., 2011) and a putative extracellular linkage component cadherin 23 (Watson et al., 2008). In the present work, we provide evidence that POU-IV regulates postmitotic differentiation of hair cells by directly activating effector genes that define mature cell identity.

In this paper, we identified the class IV POU homeodomain transcription factor (POU-IV) as an essential developmental regulator of cnidarian mechanosensory hair cells. This is the first discovery of a gene regulatory factor necessary for the development of classical mechanosensory neurons – that transmit mechanosensory information to other cells to elicit coordinated behavior – from a non-bilaterian, early-evolving animal group. Using the starlet sea anemone N. vectensis, we have shown that POU-IV is postmitotically expressed in hair cells in the ectodermal epithelium of feeding tentacles during development. In addition, we have found that null mutation of pou-iv renders the animal unable to respond to tactile stimulation to its tentacles, and results in the loss of stereovillar rootlets, but not of neurites, in hair cells. Furthermore, we have presented evidence that POU-IV binds to deeply conserved DNA sequence motifs, and directly activates the expression of a unique combination of effector genes, but not transcription factor-encoding genes, specifically in hair cells. Among the POU-IV target effector genes, we discovered the first cnidarian hair cell-specific molecular marker, polycystin 1, which encodes a transmembrane receptor of the TRP channel superfamily. The results suggest that POU-IV plays a necessary role in regulating the maturation of mechanosensory hair cells in the sea anemone by directly activating the expression of cell-type-specific effector genes. Our findings strongly support POU-IV being the terminal selector of hair cell identity in the sea anemone.

Several lines of evidence indicate that POU-IV is specifically involved in the maturation, and not progenitor proliferation, initial differentiation, or survival, of hair cells in the tentacular ectoderm in N. vectensis. First, POU-IV is postmitotically expressed in hair cells in tentacular ectoderm, and thus is unlikely to have roles in proliferation or generation of their progenitor cells. Second, in POU-IV null mutants, we have found ciliary-cone-bearing epithelial cells that resemble hair cells in morphology and position within the epithelium; these cells are characterized by having an apical cilium surrounded by a circle of microvilli, a pear-shaped cell body located in the superficial stratum of the pseudostratified epithelium, and basal neurites. None of the ciliary-cone-bearing epithelial cells express the pan-cnidocyte marker minicollagen 3, suggesting that the ciliary-cone-bearing cells in POU-IV null mutants do not represent partially differentiated nematocytes. The existence of differentiated hair cells in POU-IV null mutants implies that POU-IV is not involved in the initial differentiation of hair cells. However, the hair-cell-like cells of POU-IV null mutants failed to form a mature apical mechanosensory apparatus with stereovillar rootlets, indicating that POU-IV is essential for maturation of hair cells. Lastly, we have found no evidence that the epithelial cells expressing the non-functional form of POU-IV protein in POU-IV null mutants undergo programmed cell death in the tentacular ectoderm. Thus, POU-IV does not seem to be required for the survival of hair cells in the tentacles. Taken together, these data support the hypothesis that POU-IV regulates the maturation, but not progenitor proliferation, initial differentiation, or survival, of mechanosensory hair cells in the sea anemone.

The loss of stereovillar rootlets in hair cells in pou-iv mutants suggests that the POU-IV transcription factor regulates the expression of genes that are involved in stereovillar development. Given that stereovillar rootlets consist of actin filaments, actin-binding proteins may be regarded as potential regulators of stereovillar rootlet formation in hair cells. Among the identified POU-IV target genes expressed in hair cells is polycystin 1, which encodes a large transmembrane receptor with multiple extracellular and intracellular domains and TRP-channel-like transmembrane domains. Interestingly, its mouse homolog (PC-1) colocalizes with F-actin in inner ear hair cell stereovilli and is necessary for maintenance of stereovillar structure and normal hearing (Steigelman et al., 2011). In addition, PC-1 has been shown to regulate actin cytoskeleton reorganization in canine kidney epithelial cells (Boca et al., 2007). If the function of Polycystin 1 in modulating the organization of actin cytoskeleton is broadly conserved, N. vectensis Polycystin 1 might control the structural integrity of stereovilli in hair cells through its interaction with F-actin. POU-IV may therefore direct stereovillar development in cnidarian hair cells by activating polycystin 1. Functional analysis of N. vectensis polycystin 1 to evaluate its role in stereovillar development is warranted.

We have proposed that the lack of tentacular response to tactile stimuli in pou-iv mutants is due to the loss of structural rigidity in the apical mechanosensory apparatus – stereovilli, in particular – of hair cells, resulting from the failure of hair cells to form stereovillar rootlets. We note, however, that it could additionally be because of the functional defects in mechanotransduction channels. POU-IV is known to directly activate the expression of the mechanotransduction channel-encoding gene, mec-4, that is necessary for touch-cell function in C. elegans (Duggan et al., 1998). The Polycystin 1 protein discussed above contains transmembrane domains that are homologous to the TRP calcium channel. If this channel is involved in mechanotransduction, the loss of polycystin 1 expression in pou-iv mutants would directly lead to loss of mechanotransduction channel function. This hypothesis may be evaluated by specifically examining the role of the channel-encoding region of N. vectensis polycystin 1 in mechanotransduction.

Alternatively, the loss of polycystin 1 expression may indirectly perturb channel function. In epithelial cells of vertebrate kidneys, Polycystin 1 interacts with the calcium ion channel Polycystin 2 to form a complex that functions as a fluid flow sensor with Polycystin 1 acting as a mechanosensitive cell surface receptor and Polycystin 2 as an ion-permeant pore (reviewed in Delmas, 2004). The N. vectensis genome encodes polycystin 2 (Figure 8—figure supplement 2), which is co-expressed with polycystin 1 in the adult metacell c79 (i.e. the hair cell) (Supplementary file 9; Sebé-Pedrós et al., 2018). If these two proteins form a mechanically gated ion channel complex in hair cells as in vertebrate kidney epithelial cells, the loss of expression of polycystin 1 would perturb the ability of the complex to sense mechanical stimuli, resulting in defects in channel function. To explore this hypothesis, the important next step will be to assess whether Polycystin 1 and 2 form a complex in N. vectensis.

We note that, although our findings are consistent with the hypothesis that cnidarian hair cells function as mechanosensors, we do not rule out the possibility that cnidarian hair cells might be multimodal sensory cells; they might have additional functions as chemoreceptors and/or photoreceptors. Indeed, hair cells of the sea anemone Haliplanella luciae have been reported to respond to N-acetylated sugars by elongating their stereovilli (Mire-Thibodeaux and Watson, 1994, Watson and Roberts, 1995), indicative of chemosensory function. The metacell c79 (the presumptive hair cell) of adult N. vectensis expresses several G-protein-coupled receptor (GPCR)-encoding genes (Supplementary file 14; Sebé-Pedrós et al., 2018), some of which might encode chemosensory receptors; none were found to encode opsins. Functional analyses of these GPCRs may shed light on the molecular basis of sensory multimodality in cnidarian hair cells.

Our results indicate that the role for POU-IV in mechanoreceptor development is broadly conserved across Cnidaria and Bilateria. As mentioned above, one of the vertebrate pou-iv paralogs (Brn3c) is necessary for maturation and survival of inner ear hair cells in mice (Xiang et al., 1998; Xiang et al., 1997a, Erkman et al., 1996). Likewise, in C. elegans, differentiation of mechanosensory touch cells requires a pou-iv ortholog unc-86 (Chalfie and Sulston, 1981; Chalfie and Au, 1989; Finney and Ruvkun, 1990; Duggan et al., 1998; Chalfie et al., 1981). In Cnidaria, pou-iv is expressed in mechanosensory organs in scyphozoan and hydrozoan jellyfish (Nakanishi et al., 2010; Hroudova et al., 2012), and is necessary for differentiation of the lineage-specific mechanosensory-effector cell, the cnidocyte, in N. vectensis (Tournière et al., 2020). In this report, we have demonstrated that pou-iv has an essential role in the maturation of the classical mechanosensory neuron of Cnidaria – the concentric hair cell – using N. vectensis. These comparative data show that POU-IV-dependent regulation of mechanosensory cell differentiation is pervasive across Cnidaria and Bilateria, and likely predates their divergence. How early the role of POU-IV in mechanoreceptor differentiation emerged in animal evolution remains unresolved, and requires comparative data from placozoans and sponges, which are wanting.

We note, however, that POU-IV has a broad role in the differentiation of multiple neural cell types across Cnidaria and Bilateria. In N. vectensis, POU-IV expression is not restricted to mechanosensory hair cells and cnidocytes, but also found in RFamidergic neurons and NvElav1-positive endodermal neurons (Tournière et al., 2020). Likewise in Bilateria, POU-IV regulates the differentiation of a variety of neural cell types beyond mechanosensory cells, including chemosensory neurons in insects (Clyne et al., 1999) and photosensory neurons in vertebrates (retinal ganglion cells; e.g. Erkman et al., 1996; Gan et al., 1996). Therefore, it seems plausible that POU-IV was ancestrally involved in the differentiation of multiple neural cell types in addition to mechanosensory cells.

Interestingly, POU-IV is required for normal development of stereovilli in hair cells in both sea anemones (this study) and mice (Xiang et al., 1998), raising the possibility that POU-IV controlled the formation of the apical sensory apparatus of mechanosensory cells in the last common ancestor of Cnidaria and Bilateria, potentially via regulation of polycyctin 1. Alternatively, the essential role for POU-IV in stereovillar formation in mechanosensory cells could have evolved independently in Cnidaria and vertebrates. Comparative studies of the mechanism of stereovillar formation across sea anemones and vertebrates, along with mechanistic studies of POU-IV gene function in phylogenetically informative taxa, such as medusozoan cnidarians and acoel bilaterians, are needed to evaluate these alternative hypotheses.

Regulatory factors acting upstream of POU-IV in cnidarian hair cell development remain unknown. In Bilateria, members of the Atonal basic-loop-helix-loop-helix (bHLH) transcription factor family appear to have a conserved role in positive regulation of POU-IV expression (reviewed in Leyva-Díaz et al., 2020). For instance, vertebrate atonal genes act upstream of pou-iv genes to drive the differentiation of inner ear hair cells (Ikeda et al., 2015; Masuda et al., 2011; Yu et al., 2021) and retinal ganglion cells (Liu et al., 2001). Similarly, C. elegans atonal ortholog lin-32 controls the expression of the pou-iv ortholog unc-86 in touch sensory neurons (Baumeister et al., 1996). Although cnidarians lack unambiguous atonal orthologs, they have divergent bHLH genes that belong to the Atonal superfamily, which consists of Atonal and related bHLH gene families including Neurogenin, and NeuroD (Gyoja et al., 2012; Simionato et al., 2007). Whether these atonal-like bHLH factors function upstream of POU-IV in the context of cnidarian hair cell development needs to be assessed, as it may provide insights into the evolution of gene regulatory mechanisms underpinning mechanoreceptor development across Cnidaria and Bilateria.

In light of new comparative data reported herein emerges a model of mechanosensory cell differentiation in the last common ancestor of Cnidaria and Bilateria. We assume that the embryo of the Cnidaria-Bilateria ancestor had neurogenic ectoderm (Nakanishi et al., 2012). During late embryogenesis or postembryonic development of this ancestor, mechanosensory cell progenitors differentiated into postmitotic sensory cells in the ectoderm, extending apical cilia and basal neurites. These postmitotic sensory cells expressed the terminal selector POU-IV, which translocated to the cell nuclei, and bound to the DNA recognition motif GCAT(A/T)ATT(A/T)AT (i.e. the consensus motif across Bilateria and Cnidaria) associated with target genes in these cells. This activated the expression of effector genes, possibly including polycystin 1, whose protein products generated mechanoreceptor-specific structures necessary for mechanosensory function, such as the apical mechanosensory apparatus consisting of a cilium surrounded by a ring of stereovilli. The mature identity of the mechanosensory cell was thereby established.

Following the divergence of Cnidaria and Bilateria, POU-IV may have been recruited for the evolution of the lineage-specific mechanosensory effector – the cnidocyte – in Cnidaria, as pou-iv is essential for cnidocyte development in N. vectensis (Tournière et al., 2020). How POU-IV would have come to direct cnidocyte development remains unclear. Given that both hair cells and cnidocytes are mechanosensory cell types, it seems reasonable to expect that an ancestral POU-IV gene regulatory network that defined mechanosensory cell identity was repurposed for the emergence of cnidocytes, and should be shared across these two cell types. However, we found no evidence in support of this hypothesis; instead, our results suggest that POU-IV turns on distinct sets of genes in each cell type. One possible evolutionary scenario to account for this observation is that POU-IV initially activated the same battery of effector genes in the ancestral cnidocytes and hair cells, but POU-IV target genes diverged substantially during cnidarian evolution so that they no longer overlap between the two cell types. Another possibility is that POU-IV regulated a unique set of genes in the ancestral cnidocytes when POU-IV became part of the cnidocyte developmental gene regulatory network. This possibility seems conceivable if POU-IV expression was activated in epigenetically distinct cell lineages, so that between the cnidocyte lineage and the hair cell lineage (1) POU-IV cooperated with different co-factors, and/or (2) the accessibility of POU-IV target genes differed, which would result in differential expression of POU-IV target genes. Evidence from bilaterian models such as C. elegans and mice indicates that POU-IV cooperates with a range of different co-factors to define distinct neural identities (reviewed in Leyva-Díaz et al., 2020), suggesting an important role for POU-IV co-factors in the diversification of neural cell types. Whether evolution of POU-IV co-factors played a role in the evolution of cnidocytes remains to be tested. Investigation into the mechanism by which POU-IV activates distinct sets of genes across cnidocytes and hair cells will be the critical next step for shedding light on how POU-IV may have contributed to the evolution of the novel mechanosensory cell type of Cnidaria.

Sequencing data have been deposited in GenBank under an accession number OK338071, and in BioProject database under an accession number PRJNA767103. The scripts for RNA-Seq and ChIP-seq analysis are publicly available at https://github.com/pyrosilesl97/POU-IV_analysis (copy archived at swh:1:rev:463178242d112edab7094c12e093dd780177885b).

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Author details

1. Ethan Ozment

   Department of Biological Sciences, University of Arkansas, Fayetteville, United States

   Contribution

   Investigation, Methodology, Validation, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

2. Arianna N Tamvacakis

   Department of Biological Sciences, University of Arkansas, Fayetteville, United States

   Contribution

   Formal analysis, Investigation, Methodology, Writing - original draft

   Competing interests

   No competing interests declared

3. Jianhong Zhou

   Department of Biological Sciences, University of Arkansas, Fayetteville, United States

   Contribution

   Investigation, Methodology, Validation, Writing - original draft

   Competing interests

   No competing interests declared

4. Pablo Yamild Rosiles-Loeza

   Unidad de Genómica Avanzada (Langebio), Centro de Investigación y de Estudios Avanzados del IPN, Irapuato, Mexico

   Contribution

   Formal analysis, Investigation, Software, Visualization, Writing - original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4569-9076

5. Esteban Elías Escobar-Hernandez

   Unidad de Genómica Avanzada (Langebio), Centro de Investigación y de Estudios Avanzados del IPN, Irapuato, Mexico

   Contribution

   Formal analysis, Investigation, Software

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8892-5141

6. Selene L Fernandez-Valverde

   Unidad de Genómica Avanzada (Langebio), Centro de Investigación y de Estudios Avanzados del IPN, Irapuato, Mexico

   Contribution

   Data curation, Resources, Software, Supervision, Validation, Writing - review and editing

   Competing interests

   No competing interests declared

7. Nagayasu Nakanishi

   Department of Biological Sciences, University of Arkansas, Fayetteville, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing - review and editing

   For correspondence

   nnakanis@uark.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7516-5078

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