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During the last years, important progress has been made in modeling early brain development using 3-dimensional in vitro systems, so-called cerebral organoids. These can be grown from pluripotent stem cells of different species such as our closest living relatives, the chimpanzees and from patients carrying disease mutations that affect brain development. This offers the possibility to study uniquely human features of brain development as well as to identify gene networks altered in neurological diseases. Profiling the transcriptional landscape of cells provides insights into how gene expression programs have changed during evolution and are affected by disease. Previously, studies of this kind were realized using bulk RNA-sequencing, essentially measuring ensemble signals of genes across potentially heterogeneous populations and thus obscured subtle changes with respect to transient cell states or cellular subtypes. However, remarkable advances during the last years have enabled researchers to profile the transcriptomes of single cells in high throughput. This thesis demonstrates how single-cell transcriptomics can be used to dissect human-specific features of the developing and adult brain as well as cellular subpopulations dysregulated in a malformation of the cortex.

Eukaryotic flagella and cilia are long rod-like extensions of cells, which play a fundamental role in single cell movement, as well as in fluid transport. Flagella and cilia contain a highly evolutionary conserved mechanical structure called the axoneme. The motion of the flagellum is generated by dynein motor proteins, located all along the length of the axonemal structure. Fluorescent ATP analogs have been a useful tool to study ATPase activity of various motor proteins. \acrfull{mant} has been previously used to probe the activity of various ATPases, including dynein. It has been shown by various authors, that MANT-ATP supports dynein activity as well as the axonemal beat. However, direct observations of binding to the axonemal structure were not previously reported. Using highly sensitive fluorescent microscopy to monitor the binding of the fluorescent ATP analog, I probed dynein activity directly in the immobilized intact axoneme for the first time. To understand these kinetics a kinetic model was developed. By fitting this model to experimental data I was able to identify ATP-binding sites with distinct kinetic properties in the axoneme. I report a turnover rate of k = 0.02 s−1 at 1μM mant-ATP for dynein. Moreover, I discovered that there is binding of the ATP analog to the axoneme with a much higher rate of k = 11 s−1 at 1μM mant-ATP. By the application of this method to axonemes with reduced dyneins, it has been identified that the slow rate belongs to dynein.

Living organisms portray diverse patterns of growth and developmental regulation. The entire process, beginning from a single cell to the formation of tissues and organ systems and the final culmination in a form that characterizes a fully grown organism is closely guarded by numerous molecular pathways. Like a master conductor, the genetic material of the cell which is stored in its DNA (deoxyribonucleic acid), determines the fate of each individual cell and demarcates its developmental direction. Although in an organism, this genetic material is normally identical in every cell, there are differences in the ways cells respond to them. For example, skin cells behave differently from muscle cells and their developmental processes are highly variable in space and time. It is now known that based on intrinsic or extrinsic environmental cues, the information passed on from DNA is converted into a functional protein product through an RNA (ribonucleic acid) intermediate. Thus, different genes fire at different times leading to diverse patterns of developmental regulation among cells. However, there exists a stark contrast between the size of the genome (DNA), the transcriptome (transcribed RNAs) and the proteome (functional protein products) inside a cell. While there are abundant RNA molecules that are transcribed from the DNA, only few give rise to proteins. The search for function of RNAs that do not code for proteins is a relatively new topic in molecular biology. With advancements in sequencing methodologies, there is a rapid surge in the discovery of such molecules but due to the nonavailability of systematic tools to study them, the functional characterization of these RNAs has been relatively slow. Even among the non coding RNAs, there exists small and long varieties of which the long non coding RNAs (lncRNAs) have more heterogenous functional attributes. The roles that these lncRNAs play in development is only recently emerging, especially in the field of embryonic stem (ES) cell biology. ES cells are of particular interest to researchers due to their properties of replicating indefinitely in culture and giving rise to all the germ layers that eventually constitute an organism. These unique abilities make them perfect models to study essential cellular developmental processes and also contribute to the understanding of the molecular pathways that ultimately lead to diseases like like other processes, is orchestrated by a host of different factors in which lncRNAs are slowly emerging as important players. Although there are thousands of lncRNAs identified, only a few have been implicated in pluripotency. I reasoned that there should be more such candidates and to study them one needs to develop a strategy to functionally investigate several lncRNAs simultaneously. Loss-of-function screens have been extremely successful for dissecting the functions of protein coding genes. Among the triggers for conducting such screens, endoribonuclease-prepared small interfering RNAs (esiRNAs) have been demonstrated as effective mRNA depletion agents with minimum silencing of non-intended targets. Since these RNA interference (RNAi) agents had not been comprehensively tested on lncRNAs, I used them for conducting a screen to discover lncRNAs involved in pluripotency. Using a combination of RNAi and localization strategies, I here report the discovery of a novel lncRNA called Panct1 which through interaction with other factors takes part in the ES cell pluripotency programme. In the process of characterization of Panct1, I have also identified and partially characterized a potential DNA binding protein called CXORF23 which might emerge as an important player in the determination of stem cell fate. These discoveries hint towards the presence of more such lncRNA protein interactions and further widen our understanding of stem cell biology.

Mass spectrometry is the primary technology of proteomics. For the analysis of complex proteomes, protein identities and quantities are inferred from their peptides that are generated by cleaving all proteins with the endopeptidase trypsin. But there is one major disadvantage that is due to biophysical differences, different peptides cause different intensities. Miscellaneous approaches have been developed to circumvent this problem based on the chemical or metabolic introduction of heavy stable isotopes. This enables to monitor protein abundance differences of two or more samples on the same tryptic peptides that differ in mass only. Absolute quantification can be achieved similar by spiking-in synthetic isotopical labeled counterparts of a sample’s tryptic peptides. However, labeling technics suffer from high prices, introduced biases, need for extensive manual control, laborious implementation and implementation restrictions. Therefore, a multiplicity of label-free approaches have been developed that profit from instrumental improvements targeting reliability of identifications and reproducibility of quantitative values. No extensive systematic comparison of label-free quantitative parameters has been published so far presumably because of the laborious implementation. An analysis of primary label-free parameters and associated normalization methods is presented here that compares dynamic and linear ranges and accuracies in the estimation of protein amounts. This facilitated the establishment of label-free procedures addressing three fundamental questions in proteomics: what is a sample’s composition, are proteins that share a specific property enriched and what are the differences between two (or more) samples. A new mathematic model is presented that defines and elucidates enrichment. The procedures were applied first to analyze and compare stem cell plasma membrane proteomes. This is an ambitious model for proteomics because of only small amounts of arduous to analyze, partial hydrophobic proteins in a complex proteomic and chemical background. It is of scientific relevance, as membrane proteins are the cell’s communication interface that enable cell type specific processes and hence can be used to define, isolate and quantify those. The success of cell surface proteome enrichment, the quantitative composition of the proteome and the proteomic difference between stem cells isolated from the dental pulp and cultivated in different media is shown. Secondly, the procedures were applied to the analysis of transient protein networks that assemble onto proteo-liposomes in a newly designed recruitment assay that fully recapitulates membrane sorting as seen in vivo. All transmembrane proteins need to be trafficked to other organelles’ membranes by vesicular trafficking. Sorting signals within the cytosolic regions of the protein cargos trigger the formation of trafficking complexes around those. The transient membrane complexes additionally recognize organelle or organelle-domain specific membrane lipids, such as phosphatidylinositol phosphates. Different trafficking ways are characterized by different trafficking complexes. The elucidation of trafficking complexes that form around a transmembrane protein of interest discloses its trafficking routes and involved signaling processes. The synthetic proteo-liposomes were prepared from chemically defined lipids and heterologous expressed cytosolic domains of type I or type II membrane receptors. The proteomic analyses of such samples are challenging because of huge proteomic backgrounds of proteins binding to the liposomes irrespective of the receptor and relatively small amounts and numbers of receptor-specific binders. Though the basic idea is to elucidate sorting machineries and study membrane trafficking processes, such experiments are untargeted and miscellaneous discoveries were achieved. We elucidated that the apical determinant crumbs 2 is a cargo of the retromer complex. This revealed a fameless level of control for the establishment of cell polarity. We found retromer along with the adapter complexes AP 4 and AP 5 trafficking the beta amyloid precursor protein APP. This confirmed recent publications and yielded new insights. Moreover, many more proteins and complexes appeared to associate with the cytosolic part of APP (AICD) in a membrane context-dependent or -independent manner. Among those, some were so far unknown to interact with AICD, like mTORC1 and the PIKFyve complex.

Ligand-activated signal transduction is a process critical to cell survival and function as it serves as a means of communication between the cells and their environment. Endocytosis is generally thought to down-regulate incoming signals by reducing the surface availability of receptors. However, increasing evidence in many systems suggests a notion which is referred to as the „signalling endosome" hypothesis - that endocytosis can also actively contribute to signalling apart from clearance of activated receptors and thereby attenuation of signalling. The functional aspect of signalling endosomes has been well-characterized in several pathways including RTK and TGF-β signalling. There are, however, various other signalling pathways where the active mechanism of endocytotic regulation is yet to be understood. In this study, we probe this aspect in the cytokine signalling system, where the receptors are known to internalize but the significance of such internalization and precise mechanism is unclear. My thesis aims to elucidate the function and molecular details of internalization of cytokine receptor using interleukin-4 receptor (IL-4R) signalling as a model. IL-4 and IL-13 ligands can induce assembly of three distinct complexes: IL4 induced IL-4Rα – IL-2Rγ (type I), IL-4 induced IL-4Rα – IL-13Rα1 (type II) or the IL-13 induced IL-13Rα1-IL-4Rα (type II). The formation of any of these complexes triggers signalling through the JAK/STAT pathway. However, models of how the oligomerization of the transmembrane receptors and activation takes place are very diverse and lack a clear molecular and biophysical understanding of the underlying receptor dynamics. Previous results of the lab had shown that the affinities between subunits are low, precluding complex formation at the plasma membrane at physiological concentrations. In addition, IL-4R subunits localize in to endosomal structures adjacent to the plasma membrane. It had already been shown that the shared IL-4R subunit IL-2Rγ is internalized by a specific, actin dependent, Rac1/Pak1 regulated endocytosis route in the IL-2 context. We could show that pharmacological suppression of this endocytosis pathway also prevented IL-4 induced JAK/STAT signalling, placing endocytosis upstream of signalling. Here I show using immuno-EM techniques that these endosomal structures are multivesicular bodies. Importantly, I could show that receptor subunits are highly enriched in the limiting membrane of these endosomes relative to the adjacent plasma membrane. Using quantitative loading assays I could furthermore demonstrate that this enrichment is achieved by constitutive internalization of receptors from the cell surface into cortical endosomes. The trafficking kinetics of the receptor subunits is independent of ligand occupancy. Pharmacological inhibition shows that receptors and ligand traffic via the previously identified Rac1/Pak1 pathway. Finally, Vav2 was identified as a candidate Guanine Exchange Factor (GEF) that may regulate Rac1 activity and thereby control the actin polymerization cascade driving IL-4R endocytosis. Immunoprecipitations showed that Vav2 interacts both with the cytoplasmic tail region of the receptors and the receptor associated 2 kinase JAK3. Vav2 may thus couple the receptor/JAK complexes to the Rac1/Pak1 mediated endocytosis route. Taken together, our results suggests that stable „signalling endosomes‟ adjacent to the plasma membrane act as enrichment centres, where ligand and receptor concentrations are locally increased by constitutive trafficking. The confined environment of the endosome then compensates for the weak affinities between the ligand and receptor and facilitates ligand-mediated receptor dimerization. Importantly, overexpression of both type II IL-4R subunits renders signal transduction resistant to endocytosis inhibition, strongly suggesting that the critical factor effecting signalling is sufficient concentration, which the endosomes facilitate achieving. The endosomes are thus dispensable as signalling scaffolds when the receptors are in sufficient concentration, where activated receptors could interact with downstream pathway components. Endocytosis thus provides a crucial means for the signalling process to overcome the thermodynamic hurdles for receptor oligomerization. In conclusion, our data propose a novel, purely thermodynamic role of endosomes in regulating cytokine receptor signalling not seen in any other signalling pathway.

Cytoplasmic dynein is a ubiquitous minus-end directed motor protein that is essential for a variety of cellular processes ranging from cargo transport to spindle and chromosome positioning. Specifically, in fission yeast during meiotic prophase, the fused nucleus follows the spindle pole body in oscillatory movements from one cell pole to the other. The three molecular players that are essential to this process are: (i) the motor protein dynein, which powers the movement of the nucleus, (ii) microtubules, which provide the tracts for the movement and (iii) Num1, the anchor protein of dynein at the cortex. Dyneins that are localized to the anchor protein at the cortex and simultaneously bound to the microtubule emanating from the spindle pole body, pull on that microtubule leading to the movement of the nucleus. The spindle pole body, by virtue of its movement establishes a leading and a trailing side. Previous work by Vogel et al. has elucidated the mechanism of these oscillations as that of asymmetric distribution of dynein between the leading and trailing sides. This differential distribution is a result of the load-dependent detachment of dynein preferentially from the trailing microtubules. This self-organization model for dynein, however, requires a continuous redistribution of dynein from the trailing to the leading side. In addition, dyneins need to be bound to the anchor protein to be able to produce force on the microtubules. Anchored dyneins are responsible for many other important processes in the cell such as spindle alignment and orientation, spindle separation and rotation. So we set out to elucidate the mechanism of redistribution of dynein as well as the targeting mechanism of dynein from the cytoplasm to cortical anchoring sites where they can produce pulling force on microtubules. By employing single-molecule observation using highly inclined laminated optical sheet (HILO) microscopy and tracking of fluorescently-tagged dyneins using a custom software, we were able to show that dyneins redistributed in the cytoplasm of fission yeast by simple diffusion. We also observed that dynein bound first to the microtubule and not directly to the anchor protein Num1. In addition, we were able to capture unbinding events of single dyneins from the microtubule to the cytoplasm. Surprisingly, dynein bound to the microtubule exhibited diffusive behaviour. The switch from diffusive to directed movement required to power nuclear oscillations occurred when dynein bound to its cortical anchor Num1. In summary, dynein employs a two-step targeting mechanism from the cytoplasm to the cortical anchoring sites, with the attachment to the microtubule acting as the intermediate step.

The microphthalmia with linear skin defects syndrome (MLS) is an X-linked dominant disorder with male lethality. In the majority of the patients reported, the MLS syndrome is caused by segmental monosomy of the Xp22.3 region. To date, five male patients with MLS and 46,XX karyotype (“XX males”) have been described. Here we report on the first male case with MLS and an XY complement. The patient showed agenesis of the corpus callosum, histiocytoid cardiomyopathy, and lactic acidosis but no microphthalmia, and carried a mosaic subtle inversion of the short arm of the X chromosome in 15% of his peripheral blood lymphocytes, 46,Y,inv(X)(p22.13∼22.2p22.32∼22.33)[49]/46,XY[271]. By fluorescence in situ hybridization (FISH), we showed that YAC 225H10 spans the breakpoint in Xp22.3. End-sequencing and database analysis revealed a YAC insert of at least 416 kb containing the genes HCCS and AMELX, and exons 2–16 of ARHGAP6. Molecular cytogenetic data suggest that the Xp22.3 inversion breakpoint is located in intron 1 of ARHGAP6, the gene encoding the Rho GTPase activating protein 6. Future molecular studies in karyotypically normal female MLS patients to detect submicroscopic rearrangements including the ARHGAP6 gene as well as mutation screening of ARHGAP6 in patients with no obvious chromosomal rearrangements will clarify the role of this gene in MLS syndrome. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Eukaryotic genomes are densely compacted into chromatin, so that they can be contained in the nucleus. Despite the tight packaging genes need to be accessible for normal metabolic activities to occur, such as transcription, repair and replication. These processes are regulated by a vast number of proteins but also by the level of compaction of chromatin. The translocation of motor proteins along DNA produces torsional stress which in turn alters chromatin compaction both upstream and downstream. Few single-molecule studies have investigated the behaviour of nucleosomes when subjected to torsion. The inability to measure the applied torque though represented a major limitation to those reports. The implementation of the rotor bead assay, which allows to directly measure the torque applied in magnetic tweezers experiments, has been hindered by a difficult sample preparation procedure. In order to overcome this limitation an efficient protocol for the insertion of chemical or structural modifications in long DNA substrates was developed. This was then further expanded to allow the introduction of labels in multiple loci and/or both strands and has been used successfully in a number of studies. Furthermore this is the first report of tensile experiments performed on nucleosomes with a histone variant. H2AvD nucleosomes were studied due to the interest in the biological role of H2A.Z-family proteins. Interestingly, the variant nucleosomes appear to bind less DNA and to be evicted from the DNA at lower forces than those observed for canonical nucleosomes. These findings show an important role for the H2A-H2B dimers in the mechanical stability of nucleosomes. Furthermore these results are in agreement with recently proposed models of a dynamic nucleosome, in contrast to the long-standing view of nucleosomes as static structures.:Abstract Table of contents 1 Introduction 1.1 The transforming principle 1.2 Chromatin 1.2.1 Nucleosomes 1.2.2 The 30 nm fibre: a mirage? 1.2.3 Histone code 1.3 Histone variant H2A.Z 1.3.1 H2A.Z and transcription 1.4 Single molecule studies of chromatin 1.4.1 Chromatin under tension 1.4.2 Open nucleosome 1.4.3 Twisted chromatin 1.5 Single molecule techniques 1.5.1 Atomic force microscopy 1.5.2 Foerster resonance energy transfer 1.5.3 Magnetic tweezers 1.5.4 Worm-like chain model 2 Aims of the project 3 Cut and paste method for internal DNA labelling 3.1 Introduction 3.2 Experimental design 3.3 Results 3.3.1 Sequence design and cloning 3.3.2 Labelling and religation efficiency 3.3.3 Structural modifications 3.3.4 Labelling of multiple loci 3.3.5 Opposite-strand labelling 3.4 Discussion 4 Reconstituting chromatin 4.1 Long array of NPSs 4.1.1 Polymer physics applied to molecular cloning 4.1.2 Preventing homologous recombination 4.2 Expression and purification of histone proteins 4.2.1 Protein expression 4.2.2 Inclusions bodies 4.2.3 Histone purification 4.2.4 Octamer reconstitution and isolation 4.2.5 H2AvD 4.3 Reconstitution of nucleosomal arrays and biochemical analysis 4.3.1 Reconstitution procedure 4.3.2 Biochemical analysis 4.4 Tweezers construct with nucleosomes 5 Eviction of nucleosomes 5.1 Nucleosome eviction 5.1.1 A two-stage process 5.1.2 Chromatin fibres 5.1.3 Reassembly of nucleosomes 5.1.4 Distinct populations within nucleosome eviction events 5.1.5 Nicked and supercoilable nucleosomal arrays 5.2 Eviction of H2AvD-nucleosomes 5.2.1 H2AvD-nucleosomes bind less inner turn DNA 5.2.2 H2AvD-nucleosomes evict at lower forces 5.2.3 Likelihood of nucleosome reassembly 5.2.4 Gradual weakening of nucleosomes 5.2.5 Analysis software NucleoStep 5.3 Towards a rotor-bead assay on chromatin 5.4 Discussion 5.4.1 Nucleosome eviction in two stages 5.4.2 The fate of dimers in single molecule experiments 5.4.3 Structural origin and biological relevance of the mechanical properties of H2AvD-nucleosomal core particles 5.4.4 Monolithic or dynamic nucleosomes 6 Conclusions Bibliography Appendix 6.1 Internal labelling Procedure 6.1.1 Cloning 6.1.2 Nicking & cutting 6.1.3 The replace reaction 6.1.4 Purification 6.1.5 Ligation (optional) 6.1.6 Opposite strand labelling 6.1.7 Assessing the results of the labelling reaction 6.2 Chromatin reconstitution 6.2.1 Long array of NPSs 6.2.2 Expression and purification of histone proteins 6.2.3 Reconstitution of nucleosomal arrays and biochemical characterization 6.2.4 Simple Phenol:chloroform isolation of DNA 6.3 Magnetic tweezers experiments 6.3.1 Flow cell assembly 6.3.2 Functionalization of flow cells 6.3.3 Magnetic tweezers and rotor bead measurements 6.3.4 Force calibration List of Figures List of Tables List of publications Acknowledgements Declaration of originality

Regeneration is a process that renews damaged or lost cells, tissues, or even of entire body structures, and is a phenomenon which is widespread in the animal kingdom. Urodeles such as newts and salamanders have a remarkable regeneration ability. They can regenerate organs such as gills, lower jaws, retina, appendages like fore- and hind limbs, and also the tail including the spinal cord. The regeneration process requires the use of resident stem cells or somatic cells, which have to be reprogrammed. In both cases the reprogrammed cells are less differentiated, meaning the cell would have the ability to form any kind of fetal or adult cell which rose from the three different germ layers, the ectoderm, mesoderm and endoderm. Artificial reprogramming of differentiated mammalian somatic cell had been reported previously. It was shown that four pluripotency factors, OCT4 (also called POU5f1), SOX2, c-MYC and KLF4 are sufficient to generate an induced pluripotent stem (iPS) cell. It has been shown that some of these factors are also involved in regenerating processes. In newt limb and lens tissue, Sox2, c-Myc and Klf4 mRNA levels were upregulated in the beginning of blastema formation when compared to non-amputated tissue. Oct4 mRNA however, was not detected. During xenopus tail regeneration, Sox2 and c-Myc were expressed, while the xenopus Pou homologs Pou25, Pou60, Pou79, Pou91 were not detected. In regenerating zebrafish fin tissue, Sox2, Pou2, c-Myc and Klf4 mRNA were not upregulated. The mammalian transcription factor OCT4, a class V POU protein, is responsible in maintaining pluripotency in gastrula stage embryos. It was reported that mouse OCT4 is also expressed in the caudal node of embryos having 16 somites. It is further known that progenitors exist in mouse tailbud, which give rise to neural and mesodermal cell lineage. This suggests that the OCT4 expressing cells in caudal node might be a stem cell reservoir. Oct4 was detected in axolotl during embryonic development, and prior to my work we found Oct4 when screening the axolotl blastema cDNA library. In addition, we also identified Pou2, another class V POU gene. Phylogenetic analysis showed a clear distinction of both genes in the axolotl. We determined the mRNA pattern of Pou2 during embryogenesis and compared it to Oct4 mRNA and protein. Both genes are expressed in the primordial germ cells and the pluripotent animal cap region of the embryo. Apart from this similarity, both genes have a different expression pattern in the embryo. We are interested in the involvement of OCT4, POU2, as well as the transcription factor SOX2 in regenerating axolotl spinal cord. We asked whether the cellular pluripotent character conferred by POU factors is limited to mammals or if it is an ancient characteristic of lower vertebrates. To answer the question we performed in vitro and in vivo studies. Hence this thesis is separated into two chapter. By in vitro studies we investigated the pluripotent PouV orthologs from different species. Therefore, we performed reprogramming experiments using mouse or human fibroblasts and transduced them with axolotl Oct4 or Pou2, in combination with human or axolotl Sox2, c-Myc and/or Klf4. The generated iPS cells with the different sets of factors had similar endogenous pluripotency gene expression profiles to embryonic stem cells. Further, iPS cells expressed the pluripotency markers like OCT4, NANOG, SSEA4, TRA1-60 and TRA1-81. Another evaluation of the iPS cells was the formation of embryoid bodies. Immunouorescence staining showed that tissue from all three germ layers was formed after induction. We observed a positive staining for the endoderm marker !-FEROPROTEIN, the mesoderm marker !-SMOOTH MUSCLE ACTIN and the ectoderm marker \"III TUBULIN in the generated cells. This indicated that the iPS cells generated using axolotl Oct4 and Sox2 in combination with mammalian Klf4 and with or without c-Myc, as well as iPS cell generated with axolotl Pou2 and mammalian Sox2 and Klf4 and with or without c-Myc have a pluripotent potential. In addition, the axolotl factors are able to form heterodimers with the mammalian proteins. Furthermore, we compared the reprogramming ability with POU factors from mouse, human, zebrash, medaka and xenopus. We showed that xenopus Pou91, as the only non-mammalian example, is nearly as efficient as mouse and human Oct4 cDNAs in inducing GFP expressing cells. Also axolotl Pou2, axolotl Oct4 and medaka Pou2 showed reprogramming character however at a much lower efficiency. In contrast, zebrash Pou2 is not able to establish iPS cells. This indicates that a reprogramming ability to a pluripotent cell state is an ancient trait of Pou2 and Oct4 homologs. By in vivo studies we investigated the role of Oct4, Pou2 and Sox2 gene expression in regenerating spinal cord tissue. Performed in situ hybridizations and antibody staining studies in the regenerating spinal cord showed that Oct4, Pou2 and Sox2 were expressed during spinal cord regeneration. Knockdown experiments in regenerating spinal cord using morpholino showed that Pou2-morpholino does not have an effect. In contrast, SOX2 was required for spinal cord regeneration but to a lesser extent, than OCT4, which decreased the regenerated length signicantly compared to control. Even though, with Sox2-morpholino we did not observe the phenotype as a significantly shorter regenerated spinal cord, about 45% of SOX2 knocked down cells were not cycling and proliferating anymore. This indicates that axolotl SOX2 has an effect in regeneration. Therefore we wanted to know whether spinal cord cells would also have a pluripotent character in vivo and form other tissue types. Regenerating cells of the spinal cord are only able to form the same cell type and thus they keep their cell memory. However, when we performed transplantations of OCT4/SOX2 expressing spinal cord cells into somite stage embryos, we could show the formation of muscle cells. This shows that the spinal cord cells have the potential to change their fate in an embryonic context, where the normal environment of spinal cord has changed. However, our data do not indicate whether muscle is formed directly from the spinal cord or whether spinal cord cells fuse to developmental myoblasts, a cell type of embryonic progenitors, which give rise to muscle cells. To clearly state whether regenerating OCT4/SOX2 expressing spinal cord cells are pluripotent we have to perform OCT4 knock down in spinal cord and transplant these less proliferating cells into embryos, observing their cell fate.