Investigating alternative life history trajectories in two species of Edwardsiid sea anemones using ecological, transcriptomic, and molecular approaches

Life histories unfold within the ecological context of an organism's environment, and thus are intimately linked to organismal fitness. The evolution of alternate life history strategies, either within or between taxa, can profoundly affect ontogeny, ecology, and population dynamics. Many cnidarians (sea anemones, corals, jellyfish, etc.) exhibit complex life histories involving sexual reproduction and multiple modes of asexual reproduction. Sea anemones of the family Edwardsiidae exemplify this complexity, and are therefore an attractive system for studying the developmental and ecological ramifications of life history evolution. I used intra- and interspecific comparisons of two Edwardsiid anemones, Edwardsiella lineata, and Nematostella vectensis to investigate alternative life histories using a multifaceted approach that included field-based ecological surveys, functional genetics, transcriptomics, and phylogenetics. Both anemones are capable of sexual and asexual reproduction. N. vectensis produces a rapidly maturing direct developing larva. By contrast, E. lineata has evolved a new larval stage that parasitizes the ctenophore, Mnemiopsis leidyi. Through fieldwork surveys and laboratory culture, I documented several life history traits, such as a previously un-characterized, pre-parasitic larval stage, and the developmental dynamics of early-stage parasitic infections, that augmented gaps in our knowledge of E. lineata's life history. To better understand how and when E. lineata evolved its novel, parasitic life history, I worked with collaborators in the Finnerty lab to sequence, assemble and annotate the transcriptome. Through a multigene molecular clock approach, enabled by the E. lineata transcriptome assembly, I estimated the divergence date for these two anemones between 215-364 million years ago, thereby establishing an upper bound for the innovation of E. lineata's derived, parasitic life history. Testing a hypothesis that Wnt signaling, which patterns the oral-aboral (OA) axis during embryogenesis, also patterns the OA axis during regeneration, I demonstrated that canonical Wnt signaling is sufficient for oral tissue fate across alternate life histories (embryogenesis and regeneration) of N. vectensis. Taken together, these dissertation research activities constitute an integrative approach to investigating the evolution of life histories, and are a step towards establishing E. lineata and N. vectensis as models for studying the evolutionary developmental mechanisms of parasitism and regeneration

Mesenterial adult stem-like cells as a potential source of germinal and somatic lineages in a sea anemone

Adulte stamceller (ASC) opprettholder vekst og homeostase gjennom hele livet til dyr ved kontinuerlig å tilføre nye celler til organismen. ASC-er har blitt godt karakterisert i vertebrat-, nematode- og insektmodeller, som presenterer vevsspesifikke stamcellepopulasjoner med avstamningsbegrenset potensial (f.eks. intestinale, hematopoetiske, nevronale, epiteliale, germinale stamceller). I disse organismene er separasjonen av kimstamceller fra somatiske linjer et nøkkeltrinn under embryonal utvikling, som lenge har vært antatt å være vanlig blant metazoer. Imidlertid har studier på noen få andre bilateriske og ikke-bilateriske modellorganismer avslørt tilstedeværelsen av ASC-er som bærer både germinalt og somatisk potensial gjennom hele organismens levetid (f.eks. neoblaster i planarier, interstitielle stamceller i hydrozoiske cnidarier) Mangelen på data om ASC-er fra de fleste dyrefyla hemmer imidlertid vår forståelse av utviklingen av dyrestamceller og kimlinjesegregering. Innenfor cnidarier har ASC så langt bare blitt karakterisert i hydroziske cnidarier, som presenterer pluri- eller multipotente interstitielle stamceller (dvs. i-celler) som gir opphav til nevroner, kjertelceller, cnidocytter og gameter. Et langvarig spørsmål er om i-celler er en hydrozoa-spesifikk egenskap eller fra forfedre til cnidaria. I oppgaven min har jeg som mål å bidra til vår forståelse av utviklingen av cnidaria og dyrestamceller ved å identifisere og karakterisere ASCs i en antozoisk cnidarie, sjøanemonen Nematostella vectensis. For å finne ASC-er i Nematostella, undersøkte jeg det romlige uttrykket av konserverte markørgener for kimlinje- og multipotens ortologer (f.eks. piwi, vasa) i juvenile og voksne polypper, og utførte avstamningssporing og enkeltcellet RNA-sekvensering (scRNA-seq) for å avdekke deres molekylære profiler og potensialer. Ved å kombinere og analysere Vasa2 immunfarging med vasa2 og piwi1 transgene reporterlinjer i unge og voksne, karakteriserte jeg en populasjon av mesenteriale, ekstragonadale Vasa2+/Piwi1+ stam-lignende celler og deres gonadale Vasa2+/Piwi1+ germinal avkom. Påfallende nok fant jeg også at Vasa2+/Piwi1+ stam-lignende celler gir opphav til rikelig med gastrodermale celler av somatiske avstamning, delvis bestående av soxB(2)-uttrykke neuronale stamceller. Ved å bruke scRNA-seq på vevsanrikede prøver, var jeg i stand til å identifisere celleklynger med delvis overlappende ekspresjonsprofiler tilsvarende (1) mesenterial Vasa2+/Piwi1+ stam-lignende cellepopulasjon, (2) meiotisk oogonia, (3) celler i M -fase, (4) soxB(2)+ nevrale forløpere og (5) cnido-glandulære traktat-forløpere. Til sammen antyder resultatene mine at den mesenteriale Vasa2+/Piwi1+ stam-lignende celle-populasjonen består av multipotente ASC-er med både germinalt og somatisk (inkl. neuronalt) potensial. Siden slike celler sannsynligvis bidrar til vekst, vevs-homeostase og reproduksjon under unge og voksenstadier i Nematostella-polypper, åpner oppgaven min for en bedre karakterisering av de cellulære prosessene som ligger til grunn for helkropps-regenerering, aseksuell reproduksjon og plastisitet i kroppsstørrelse hos cnidarier. Det delte potensialet, uttrykket av genetiske markørene og cellulære funksjonene med hydrozoa i-celler, fører oss til å foreslå at Vasa2+/Piwi1+ ASC-er med blandet germinalt og somatisk potensiale er en egenskap fra forfedre for cnidarier.Adult stem cells (ASCs) sustain growth and homeostasis throughout the lifetime of animals by continuously supplying new cells to the organism. ASCs have been well characterized in vertebrate, nematode and insect models, which present tissue-specific stem cell populations with lineage-restricted potentials (e.g. intestinal, hematopoietic, neuronal, epithelial, germinal stem cells). In these organisms, the separation of germline stem cells from somatic lineages is a key step during embryonic development, which has long been thought to be common among metazoans. However, studies on a few other bilaterian and non-bilaterian model organisms have revealed the presence of ASCs bearing both germinal and somatic potentials throughout the lifetime of the organism (e.g. neoblasts in planarians, interstitial stem cells in hydrozoan cnidarians). The lack of data on ASCs from most animal phyla however hampers our understanding of the evolution of animal stem cells and germline segregation. Within cnidarians, ASCs have so far only been characterized in hydrozoan cnidarians, which present pluri- or multipotent interstitial stem cells (i.e. i-cells) that give rise to neurons, gland cells, cnidocytes and gametes. A longstanding question is whether i-cells are a hydrozoan-specific trait or ancestral to cnidarians. In my thesis, I aim to contribute to our understanding of cnidarian and animal stem cell evolution by identifying and characterizing ASCs in an anthozoan cnidarian, the sea anemone Nematostella vectensis. To find ASCs in Nematostella, I investigated the spatial expression of conserved germline and multipotency marker gene orthologs (e.g. piwi, vasa) in juvenile and adult polyps, and performed lineage tracing and single-cell RNA sequencing (scRNA-seq) to unveil their molecular profiles and potentials. By combining and analysing Vasa2 immunostaining with vasa2 and piwi1 transgenic reporter lines in juveniles and adults, I characterized a population of mesenterial, extra-gonadal Vasa2+/Piwi1+ stem-like cells and their gonadal Vasa2+/Piwi1+ germinal progeny. Strikingly, I found that Vasa2+/Piwi1+ stem-like cells also give rise to abundant, gastrodermal somatic progeny cells, partially consisting of soxB(2)-expressing neuronal progenitor cells. Using scRNA-seq on tissue-enriched samples, I was able to identify cell clusters with partially overlapping expression profiles corresponding to (1) the mesenterial Vasa2+/Piwi1+ stem-like cell population, (2) meiotic oogonia, (3) cells in M-phase, (4) soxB(2)+ neural progenitors and (5) cnidoglandular tract progenitor cells. Altogether, my results suggest that the mesenterial Vasa2+/Piwi1+ stem-like cell population consists of multipotent ASCs with both germinal and somatic (incl. neuronal) potential. As such cells likely contribute to growth, tissue homeostasis and reproduction during juvenile and adult stages in Nematostella polyps, my thesis opens the door to a better characterisation of the cellular processes underlying whole-body regeneration, asexual reproduction, and body size plasticity in cnidarians. The shared potential, expression of genetic markers and cellular features with hydrozoan i-cells lead us to propose that Vasa2+/Piwi1+ ASCs with mixed germinal and somatic potential are an ancestral trait of cnidarians.Doktorgradsavhandlin

Mechanisms of epithelial morphogenesis and integrity during Nematostella vectensis development and Shigella pathogenesis

The transition to animal multicellularity involved the evolution of single cells organizing into sheets of tissue. The advent of tissues allowed for specialization and diversification, which led to the formation of complex structures and a variety of body plans. These epithelial tissues undergo morphogenesis during animal development, and the establishment and maintenance of their polarity and integrity is crucial for homeostasis and prevention of pathogenesis. This architecture is dynamically maintained through a variety of cellular processes including the regulation of intracellular transport, cytoskeletal modulation, and cell adhesion. While studies in established model organisms and cell culture have contributed to our current knowledge of these processes, evolutionary and in vivo perspectives are largely lacking. Our efforts to gain a better understanding of epithelial biology have centered around two main themes: 1) Ancient mechanisms of morphogenesis during animal development and 2) Modulation of epithelial architecture during pathogenesis. First, to address the ancient mechanisms of epithelial morphogenesis, we examine tentacle development in the cnidarian Nematostella vectensis as a model of outgrowth formation. Through drug treatments, transcriptional analysis and imaging experiments, our study identifies molecular and cellular mechanisms that act during elongation of the tentacles and body column. At the onset of tentacle development, we observe an ectodermal placode that forms at the oral end of the animal, which is transcriptionally patterned into four tentacle buds. Subsequently during morphogenesis, our results show that cell shape changes and cell rearrangements act during elongation of the bud into a mature tentacle. In the body column during elongation, we also observe a period of oriented cell divisions along the oral-aboral axis. Together, our results reveal ancient cellular and molecular mechanisms of epithelial morphogenesis during development in an early-branching metazoan. Second, to explore alterations in epithelial architecture and integrity during bacterial pathogenesis, we express a Shigella bacterial virulence protein, VirA, in Drosophila and vertebrate tissue. Previous reports on the function of VirA have only employed in vitro and cell culture assays, so the function of VirA in an epithelial context remains largely unknown. Through in vivo expression and imaging experiments, we show that VirA expression in Drosophila disrupts epithelial architecture and cell polarity, with no discernible effects on microtubule stability. In the Drosophila salivary gland and eye imaginal disc, cells expressing VirA round and lose polarity markers. We observe a similar apical cell rounding phenotype when VirA is expressed in chick neural tube, implying a conserved mechanism of VirA function in vertebrates. Finally, we demonstrate a mislocalization of Rab11 in VirA expressing epithelia, suggesting a potential defect in vesicle trafficking. Taken together, our results reveal a novel function for VirA in disruption of cell polarity or adhesion, possibly through vesicle trafficking, leading to a breakdown of epithelial integrity facilitating the pathogenesis of Shigella in the human intestinal epithelium

Inhibition of Runx by Ro5-3335 Affects Nematostella Regeneration

This undergraduate research project is being made available in KU ScholarWorks with the permission of the author. The project was supervised by Dr. Paulyn Cartwright, associate professor of Ecology and Evolutionary Biology at the University of Kansas.The sea anemone, Nematostella vectensis, has the ability to fully regenerate amputated body parts. We hypothesize that Runx, a transcription factor, controls the cellular processes for regeneration, specifically the transition between cellular proliferation and cellular differentiation. A known inhibitor to the Runx pathway is Ro5-3335, a benzodiazepine. Inhibiting the Runx pathway by Ro5-3335 will help determine if Runx is necessary for proper regeneration in Nematostella. We introduced bisected Nematostella polyps to Ro5-3335 for a 24-48 period and observed regeneration of oral ends. Tentacle regeneration appeared delayed in treated polyps compared to the controls. It was not until three weeks post-treatment that the treated animals recovered normal regeneration. We conclude that Ro5-3335 appears to repress regeneration in the polyps which suggests that the Runx pathway is important for proper regeneration in Nematostella

Specification and differentiation of neural cells in Nematostella vectensis

Postponed access: the file will be accessible after 2022-02-21During embryonic development, early neurogenesis can be divided into several components, such as the origin, proliferation and movement of neural stem cells and progenitor cells, which are regulated by conserved genes and signalling pathways. These fundamental aspects of neurogenesis have been extensively studied in only a few bilaterian model organisms, leaving many questions regarding the evolution of this process open. The cnidarian and bilaterian lineages are sister groups that separated approximately 600 million years ago. Cnidarians have an informative position to study the early evolution of cellular and molecular aspects of neurogenesis and to understand common principles of neural development. Nematostella vectensis is a sea anemone, member of the phylum Cnidaria. They possess epithelial neural progenitor cells that express NvSoxB(2) and Atonal-like transcription factors. The Notch signalling pathways regulates the number of progenitor cells and achaete-scute is involved in further development. While some aspects of neural progenitor cells have been identified, little is known regarding the specification and differentiation of neural subtypes. The present thesis focuses on those aspects of neurogenesis. Through a candidate gene approach, two transcriptions factors were selected for further analysis. Expression analysis and generation of a transgenic reporter line for the single POU class 4 gene in Nematostella vectensis, revealed that this gene is expressed in diverse post-mitotic neural cell types. I analysed its function by first generating a mutant line with CRISPR/Cas9 and secondly by analysing and comparing transcriptomes derived from the mutants and from different neural cell populations. This study shows that NvPOU4 is involved in the terminal differentiation program of different neural cells, a function conserved with many bilaterians. I further discuss the relevance of POU4 genes, and terminal selectors in general, for studying the evolution of cell types in metazoans. The second candidate gene involved in neural differentiation is Insulinoma associated 1 (Insm1). Using expression analysis and a transgenic reporter line, I show that NvInsm1-expressing cells give rise to sensory and ganglion neurons as well as to gland cells. In this study, I demonstrate that those cell types originate from a population of progenitor cells expressing NvSoxB(2). I further discuss the implications of these results regarding the developmental and evolutionary origin of neural and gland cells in metazoans. The new findings and molecular tools generated in this thesis provide the foundation for a better understanding of evolutionary and developmental aspects of nervous system formation

The dystrophin-glycoprotein complex in the development of Nematostella vectensis: Expression analyses and generation of a mutant by genome editing

Postponed access: the file will be accessible after 2022-12-03The development and function of cells in the nervous system and the musculature require regulated interactions with the extracellular matrix. The dystrophin-glycoprotein complex is an important regulator of these interactions. A central component of this complex is dystroglycan, a membrane protein that is heavily glycosylated on its extracellular part. This glycosylation is initiated by O-mannosyltransferases and is then expanded by a series of additional glycosyltransferases. The sea anemone Nematostella vectensis represents an evolutionary ancient group of animals and has emerged as a powerful tool to study neurogenesis and development in an evolutionary context. The genome of Nematostella encodes all members of the dystrophin-glycoprotein complex, introducing the question of their role in this animal. In this study, we show that the expression pattern of dystroglycan and five glycosyltransferases are compatible with different roles of dystroglycan O- mannosylation. By in-situ hybridization, we identified POMGNT1, POMT1 and POMT2 to display a similar expression pattern throughout early development. Double fluorescence in- situ hybridization shows the enzymes to be co-expressed with each other, suggesting a shared function. Difference in early expression between the glycosyltransferases and dystroglycan might indicate a dystroglycan-independent function of O-mannosylation, whereas the similar expression in later stages indicate a potential role for dystroglycan-dependent O-mannosylation during Nematostella development. Furthermore, double fluorescence in-situ hybridization found POMGNT1 to be expressed in FoxQ2d-expressing sensory cells, suggesting the enzyme to have a role in sensory cells. Finally, CRISPR/Cas9 was successfully used to introduce a mutation in the gene of POMGNT1, to further study the function of this glycosyltransferase and its substrate in Nematostella development. We anticipate this study to be a starting point for future studies to provide insight on the role of the dystrophin- glycoprotein complex and the necessity of alpha-dystroglycan O-mannosylation on the nervous and muscular systems of Nematostella vectensis.Masteroppgave i molekylærbiologiMOL399MAMN-MO

Unveiling the Ancestral Function of a Neuroendocrine Regulator, POU-I/Pit1: Insights from Gene Expression Analysis in the Sea Anemone Nematostella vectensis

Cnidaria (i.e., sea anemones, jellyfish, corals) and Bilateria (i.e., vertebrates, sea stars, fruit flies), are sister groups that diverged around 600 million years ago. Despite the long evolutionary time, many cellular differentiation mechanisms, cell types, tissues and behaviors are conserved. Such as neurons, mechanosensory hair cells, feeding behaviors, peristaltic movements, and sleep. Recent advances in genomics, molecular biology and microscopy have fueled an increased interest in understanding cnidarian nervous and neuroendocrine systems. Understanding the developmental mechanisms and the mode of operation of Cnidarian nervous systems helps to reconstruct the ancestral nervous system of the last common ancestor of Cnidaria and Bilateria. Thus, also shedding light in fundamental aspects of Bilaterian nervous systems. Here, the ‘starlet sea anemone’ Nematostella vectensis, a powerful cnidarian model organism was used to address the gene expression pattern of Pit1, a conserved gene shared between Cnidaria and Bilateria. In Chapter 1, a method to extract DNA and genotype embryos of Nematostella without sacrificing the animal was established, with possible application to other non-sea anemone cnidarians. Early genotyping is fundamental for addressing phenotypes during development, thus opening the door to study the function of any gene of interest during larval pre-metamorphic stages. In Chapter 2, the expression pattern of Pit1 and detailed cellular morphology of Pit1-positive cells was characterized for the first time in Nematostella. Complex neuronal networks and diverse sensory cells were found. Furthermore, the foundation for future functional studies of Pit1 was laid by establishing stable CRISPR-Cas9 knockout and transgenic reporter lines

Unveiling the Ancestral Function of a Neuroendocrine Regulator, POU-I/Pit1: Insights from Gene Expression Analysis in the Sea Anemone Nematostella vectensis

Cnidaria (i.e., sea anemones, jellyfish, corals) and Bilateria (i.e., vertebrates, sea stars, fruit flies), are sister groups that diverged around 600 million years ago. Despite the long evolutionary time, many cellular differentiation mechanisms, cell types, tissues and behaviors are conserved. Such as neurons, mechanosensory hair cells, feeding behaviors, peristaltic movements, and sleep. Recent advances in genomics, molecular biology and microscopy have fueled an increased interest in understanding cnidarian nervous and neuroendocrine systems. Understanding the developmental mechanisms and the mode of operation of Cnidarian nervous systems helps to reconstruct the ancestral nervous system of the last common ancestor of Cnidaria and Bilateria. Thus, also shedding light in fundamental aspects of Bilaterian nervous systems. Here, the ‘starlet sea anemone’ Nematostella vectensis, a powerful cnidarian model organism was used to address the gene expression pattern of Pit1, a conserved gene shared between Cnidaria and Bilateria. In Chapter 1, a method to extract DNA and genotype embryos of Nematostella without sacrificing the animal was established, with possible application to other non-sea anemone cnidarians. Early genotyping is fundamental for addressing phenotypes during development, thus opening the door to study the function of any gene of interest during larval pre-metamorphic stages. In Chapter 2, the expression pattern of Pit1 and detailed cellular morphology of Pit1-positive cells was characterized for the first time in Nematostella. Complex neuronal networks and diverse sensory cells were found. Furthermore, the foundation for future functional studies of Pit1 was laid by establishing stable CRISPR-Cas9 knockout and transgenic reporter lines

Food uptake, lipid transport and vitellogenesis in the sea anemone Nematostella vectensis

Balancing energy input and output is crucial for the survival of all organisms, and involves the coordination of many physiological processes such as food uptake, nutrient storage, reproduction and growth. The uptake of food particles through endocytic mechanisms (e.g. phagocytosis, receptor-mediated endocytosis) is broadly observed and likely the ancestral mode of feeding in metazoans. However, only little is known about the biology and evolution of endocytic cell types involved in animal nutrition. Similarly, the dynamics and molecular pathways underlying the transport of nutrients is poorly investigated in animals without a circulatory system. The lack of available studies, especially in non-bilaterian animals (e.g. cnidarians, sponges) leaves a number of key questions unresolved: how did endocytic cell types evolve? What are the ancestral modalities of nutrient transport in animals? In my thesis, I address these questions by investigating the cells and molecular pathways underlying food uptake and, as a specific example of lipid transport, vitellogenesis, in the sea anemone Nematostella vectensis. By characterizing the path of food particles and dietary lipids from their ingestion to their incorporation into yolk, I aim to fill in gaps in our understanding of nutrient uptake and transport in non-bilaterians and thereby shed light on the evolution of these processes in animals. In Nematostella, nutrient acquisition starts with the extracellular digestion of prey in the gastric cavity through secreted digestive enzymes. Using single-cell RNA sequencing (scRNA-seq), I characterized the cellular composition of the gastrodermal folds lining the gastric cavity (mesenteries) and found a high diversity of specialized gland cells expressing specific enzymatic repertoires. Extra-cellular digestion is followed by endocytosis and subsequent intracellular digestion of food particles. By using particle uptake assays, I revealed that phagocytosis and receptor-mediated endocytosis predominantly occur in specific regions of the mesenteries in Nematostella, highlighting a surprising regionalization of the anthozoan gastrodermis. These regions colocalize with the cellular expression of Nematostella orthologs of bilaterian genes typically involved in endocytosis (e.g. mannose receptor, clathrin). This strongly supports the digestive function of these cells and indicates a conserved nature of endocytic molecular pathways between cnidarians and bilaterians. These results were further validated by scRNA-seq, which revealed three distinct populations of trophic endocytes co-localizing within the endocytic region of the mesentery. In bilaterians, dietary nutrients are most often transported towards other tissues via the circulatory system in order to be stored or to support the metabolism of peripheral tissues. Cnidarians lack a circulatory system, and the gastro-vascular cavity is thought to distribute nutrients throughout the body. The extracellular matrix (mesoglea) was previously proposed to participate in nutrient transport, but its role in this process has so far been unclear. In the present work, I describe for the first time the dynamic trans-epithelial transport of lipids from the gastric cavity into maturing oocytes located in the mesoglea in a cnidarian. Consistent with their function in shuttling lipids between the gastric cavity and the oocyte, somatic cells of the gonad epithelium also produce the glycolipoprotein Vitellogenin, a conserved yolk precursor. Gene expression data shows that the uptake of Vitellogenin into growing oocytes likely occurs through receptor-mediated endocytosis using orthologs of the vldlr/apolipophorin receptor gene family. This supports the hypothesis that a specific Vitellogenin ligand/receptor pair is highly conserved in vitellogenesis between cnidarians and bilaterians. Finally, I characterized the expression and protein localization of ApoB, a Nematostella ortholog of the bilaterian systemic lipid transport proteins Apolipoprotein-B/Apolipophorins. ApoB protein was not detected in growing oocytes in Nematostella but surprisingly localized in spermaries, suggesting a role during spermatogenesis. Overall, these results demonstrate the mesogleal transport of lipids potentially using conserved lipoprotein-lipoprotein receptor pairs in the absence of a circulatory system, and raise the possibility of a rudimentary systemic lipid transport system in Nematostella. Altogether, my thesis revealed that nutrient uptake in Nematostella involves a remarkable diversity of specialized cell types that define functional domains in the mesenteries. The molecular machinery for food uptake, intracellular digestion and lipid transport seems to be highly conserved between Nematostella and bilaterians, providing an opportunity to elucidate the ancestral state of mechanisms underlying energy homeostasis in the last common ancestor to cnidarians and bilaterians.Doktorgradsavhandlin

Expression and function of the transcription factor Rx in Nematostella vectensis

The development of the central nervous system is highly studied and a comparatively well understood field of neurogenesis. The fundamental aspects of neurogenesis have however mostly been studied in a select few bilaterian model organisms, leaving many questions regarding the evolution of this process open. The cnidarian and bilaterian lineages are sister groups that separated approximately 600 million years ago. Cnidarians occupy an informative phylogenetic position to study the early evolution of cellular and molecular aspects of neurogenesis, and to understand common principles of neural development. The Cnidaria sea anemone Nematostella vectensis has in recent years emerged as one of the most tractable cnidarian models for the study of neurogenesis and development in an evolutionary context. The later steps of neural differentiation in Nematostella are yet to be fully understood. Neuronal markers have been found for different neural sub-populations, one of which is the transcription factor NvFoxQ2d that gives rise to sensory cells in Nematostella. Although the function of NvFoxQ2d is yet to be determined, studies are being performed to further understand this transcription factor, one of which is a transcriptome of the fluorescent cells in the NvFoxQ2d::mOrange transgenic reporter line. During the analysis of this dataset, the transcription factor NvRx was found to be highly upregulated in this cell population. While the Retinal homeobox gene (Rx) has a well-described role in the formation of the retina, and the formation and proliferation of retinal progenitor cells in vertebrates, the function of NvRx in the eyeless Nematostella is in contrast yet to be determined. By performing a double fluorescence in-situ hybridization, we show that NvRx is expressed in the majority of the NvFoxQ2d cell population. We hypothesised that NvRx could play a role in the regulation of these NvFoxQ2d-expressing cells, and by performing an in-situ on embryos injected with NvRx shRNA injection, we show that NvRx acts as an upstream regulator of NvFoxQ2d. We also found NvRx-expressing cells in close proximity to NvElav1-expressing neural cells, indicating that these cells could be descended from the same progenitor cells. Finally, a CRISPR/Cas9 mediated mutant line was successfully generated to further study the function of NvRx in Nematostella developmentMasteroppgave i molekylærbiologiMOL399MAMN-MO