Cephalopods provide an unprecedented opportunity for comparative studies of the developmental genetics of organ systems that are convergent with analogous vertebrate structures. The Sox-family of transcription factors is an important class of DNA-binding proteins that are known to be involved in many aspects of differentiation, but have been largely unstudied in lophotrochozoan systems. Using a degenerate primer strategy we have isolated coding sequence for three members of the Sox family of transcription factors from a cephalopod mollusk, the European cuttlefish Sepia officinalis: Sof-SoxE, Sof-SoxB1, and Sof-SoxB2. Analyses of their expression patterns during organogenesis reveals distinct spatial and temporal expression domains. Sof-SoxB1 shows early ectodermal expression throughout the developing epithelium, which is gradually restricted to presumptive sensory epithelia. Expression within the nervous system appears by mid-embryogenesis. Sof-SoxB2 expression is similar to Sof-SoxB1 within the developing epithelia in early embryogenesis, however appears in largely non-overlapping expression domains within the central nervous system and is not expressed in the maturing sensory epithelium. In contrast, Sof-SoxE is expressed throughout the presumptive mesodermal territories at the onset of organogenesis. As development proceeds, Sof-SoxE expression is elevated throughout the developing peripheral circulatory system. This expression disappears as the circulatory system matures, but expression is maintained within undifferentiated connective tissues throughout the animal, and appears within the nervous system near the end of embryogenesis. SoxB proteins are widely known for their role in neural specification in numerous phylogenetic lineages. Our data suggests that Sof-SoxB genes play similar roles in cephalopods. In contrast, Sof-SoxE appears to be involved in the early stages of vasculogenesis of the cephalopod closed circulatory system, a novel role for a member of this gene family.

SOX14 is a member of the SOXB2 subgroup of transcription factors implicated in neural development. Although the first SOX14 gene in vertebrates was cloned and characterized more than a decade ago and its expression profile during development was revealed in various animal model systems, the role of this gene during neural development is largely unknown. In the present study we analyzed the expression of SOX14 in human NT2/D1 and mouse P19 pluripotent embryonal carcinoma cells. We demonstrated that it is expressed in both cell lines and upregulated during retinoic acid induced neural differentiation. We showed that SOX14 was expressed in both neuronal and non-neuronal differentiated derivatives, as revealed by immunocytochemistry. Since it was previously proposed that increased SOXB2 proteins level interfere with the activity of SOXB1 counteracting partners, we compared expression patterns of SOXB members during retinoic acid induction of embryonal carcinoma cells. We revealed that upregulation of SOX14 expression is accompanied by alterations in the expression patterns of SOXB1 members. In order to analyze the potential cross-talk between them, we generated SOX14 expression construct. The ectopic expression of SOX14 was demonstrated at the mRNA level in NT2/D1, P19 and HeLa cells, while an increased level of SOX14 protein was detected in HeLa cells only. By transient transfection experiments in HeLa cells we showed for the first time that ectopic expression of SOX14 repressed SOX1 expression, whereas no significant effect on SOX2, SOX3 and SOX21 was observed. Data presented here provide an insight into SOX14 expression during in vitro neural differentiation of embryonal carcinoma cells and demonstrate the effect of its ectopic expression on protein levels of SOXB members in HeLa cells. Obtained results contribute to better understanding the role of one of the most conserved SOX proteins.

Neurogenesis is the generation of neurons from stem cells, a process that is regulated by SoxB transcription factors (TFs) in many animals. Although the roles of these TFs are well understood in bilaterians, how their neural function evolved is unclear. Here, we use Hydractinia symbiolongicarpus, a member of the early-branching phylum Cnidaria, to provide insight into this question. Using a combination of mRNA in situ hybridization, transgenesis, gene knockdown, transcriptomics, and in vivo imaging, we provide a comprehensive molecular and cellular analysis of neurogenesis during embryogenesis, homeostasis, and regeneration in this animal. We show that SoxB genes act sequentially at least in some cases. Stem cells expressing Piwi1 and Soxb1, which have broad developmental potential, become neural progenitors that express Soxb2 before differentiating into mature neural cells. Knockdown of SoxB genes resulted in complex defects in embryonic neurogenesis. Hydractinia neural cells differentiate while migrating from the aboral to the oral end of the animal, but it is unclear whether migration per se or exposure to different microenvironments is the main driver of their fate determination. Our data constitute a rich resource for studies aiming at addressing this question, which is at the heart of understanding the origin and development of animal nervous systems.