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The success rate of regenerative medicine largely depends on the type of stem cells applied in such procedures. Consequently, to achieve the needed level for clinical standardization, we need to investigate the viability of accessible sources with sufficient quantity of cells. Since the oral region partly originates from the neural crest, which naturally develops in niche with decreased levels of oxygen, the main goal of this work was to test if human oral mucosa stem cells (hOMSC) might be used to treat neurons damaged by anoxia. Here we show that hOMSC are more resistant to anoxia than human induced pluripotent stem cells and that they secrete BDNF, GDNF, VEGF and NGF. When hOMSC were added to human neurons damaged by anoxia, they significantly improved their survival. This regenerative capability was at least partly achieved through miR-514A-3p and SHP-2 and it decreased in hOMSC exposed to neural cells for 14 or 28 days. In addition, the beneficial effect of hOMSC were also confirmed in mice affected by stroke. Hence, in this work we have confirmed that hOMSC, in a time-limited manner, improve the survival of anoxia-damaged neurons and significantly contribute to the recovery of experimental animals following stroke.

Objectives: To describe neuronal differentiation from the disomic (D21) and trisomic (T21) isogenic human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs) derived from persons with partial and full trisomy 21, and mosaic Down syndrome (DS). Methods: NSC were generated form the isogenic iPSCs following published protocol (1) and expanded in our laboratory. NSCs, between passage 5 and 10 were seeded on the commercially available mouse astrocytes and differentiated to the mature neurons 100 days in vitro (DIV) (2). Moreover, cerebral organoids were generated following published protocol (3) with our modification (4) and fixed at 30, 50, 70 and 100 DIV and analysed by immunohistochemistry. Results: In our previous work we published neuronal differentiation from EBs D3 and 47-1 (5). Here we find that Tubb3 positive cells show a specific neuronal phenotype. Disomic cells showed normal neuronal morphology, on the other hand trisomic cells showed a very specific cellular dysmorphology: neurons were smaller, shorter with thicker processes, and abnormal branching patterns. The same pattern we have observed in neurons derived form iPSCs in monolayer as well as in cerebral organoids. Both, neurons in monolayer, as well as in cerebral organoids expressed all neuronal markers: pan neuronal markers (Dcx, MAP2. Tubb3, SMI, 3R-Tau) and cortical layer specific markers (Reelen, Brn2, Ctip2, SATB2, TBR1 and TBR2). Conclusion: Trisomic cells could differentiate to mature, synaptic active neurons but morphology of trisomic cells shows a unique and specific dysmorphology pattern.

Human, mammalian, brain development is a very complex process and starts shortly after gastrulation. Human in vitro models for brain development as well as pathological changes were published in last few years, eliminating the ethical concerns that accompany the study of aborted foetal material. One of the recent approaches is the development of induced pluripotent stem cell (iPSC) technology and different types of organoids, which can be applied to any species. In our previous work we developed iPSCs from an adult individual with constitutional mosaicism for Down Syndrome (DS) (1). From those iPSCs, cerebral organoids were grown and corticogenesis and Alzheimer-like pathology in vitro were studied (2). Mixed cerebral (2), as well as midbrain (unpublished) organoids were grown in vitro from the isogenic, trisomic and disomic iPSCs. Organoids were analysed histologically by IMARIS software. Foetal human brains were stained with the same combinations of antibodies and the data were compared. Anonymized post-mortem brain samples were obtained from the Brain Bank of the Croatian Institute for Brain Research (CIBR). Isogenic cerebral organoids expressed the markers of all six cortical layers (Reelin, FOXG1, SATB2, TBR1, Ctip2 and Brn2) after 100 days differentiation in vitro (DIV). At the early time point of DIV30 the majority of cells were Nestin, SOX2, PAX6 positive. At that time point neurogenesis had already started, so we observed TRBR2, DCX, MAP2 and TUBB3 positive cells in disomic and trisomic organoids. Following further neuronal differentiation of cerebral organoids we observed decreased number of neural stem cells and increased number of neuronal markers specific for all six layers. On the other hand, midbrain organoids expressed markers specific for midbrain. At the early time points midbrain organoids expressed neuronal stem cells markers, similar to cerebral organoids, but with organoid maturation we found midbrain specific cells such as NURR1 and OTX2 specific for the early stages, and later Dopamine β Hydroxylase, NET and TH. Finally, all those markers were compared with expression in foetal human brain from individuals with Down Syndrome as well as euploid controls age, sex and brain region matched. Our data showed that isogenic cerebral organoids grown from iPSCs are good tool for multidisciplinary studies. In our group we study corticogenesis, synaptogenesis, pathology and possible drug treatments.

Background: Specific cell types from people with Down syndrome (DS) show faster accumulation of DNA damage and epigenetic senescence marks. Causative mechanisms remain un-proven ranging from amplified chromosomal instability to actions of chromosome 21 genes. Plasma immunoglobulin G (IgG) glycosylation profiles are established as a reliable predictor of biological and chronological ageing. Methods: We performed IgG glycan profiling of n=246 individuals with DS (208 adults and 38 children) clinically characterised for co- morbidities, from three European countries, and compared these to age-, sex- and demography- matched general populations. Results: We uncovered very significantly increased IgG glycosylation ageing marks associated with DS, beginning in early childhood. Average levels of IgG glycans without galactose (G0) and those with two galactoses (G2) as a function of age in persons with DS corresponded to levels detected in 19 years older euploid individuals. Sub-cohorts of DS without any known co-morbidities from different countries showed a similar result, eliminating DS- linked autoimmune diseases, frequent infections, or Alzheimer’s dementia as indirect causes of accelerated biological ageing. Importantly, no difference in slope of the curve relating IgG- glycan-age to chronological age was observed for any of the 3 analysed populations, discouraging the amplified-chromosomal-instability explanation. Remarkably, IgG-glycan profiles of a child with DS caused by a short segmental duplication of only 31 genes on chromosome-21 (DS-critical-region) had values like age-matched whole-chromosome-trisomy- 21 children, pointing to a causative contribution of one or more of the 31 genes. Conclusion: This shifts the paradigm from an (essentially untreateable) “amplified instability” mechanism, to a specific gene-overdose-driven cause of a progeria-like syndrome, opening possibilities for therapeutic amelioration strategies. We generated an induced pluripotent stem cell (iPSC) model of this child with partial trisomy 21, and editing experiments using CRISPR-Cas9 are underway in order to dissect the causative overdosed genes and mechanisms responsible for the accelerated ageing in DS.

The goal was to explore the difference in N- glycosylation between euploid disomic (D21) and trisomic (T21) isogenic human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs) derived from person with mosaic Down syndrome (DS) trisomy of 21st chromosome.....

Synaptic glycoprotein neuroplastin (Np) is a crucial synaptic junction (SJ) proteome member. Evidenced functions of Np include regulating Ca2+ clearance and its involvement in long-term potentiation. We have previously reported differential immunohistochemical pattern of Np across brain regions in Toll-like receptor 2 (TLR2) knock-out (KO) mice and assumed that detected alterations might accompany large-scale changes in the synaptic environment in TLR2KO mice. These findings guided us on a journey to address whether the absence of TLR2 affects the SJ proteome. The study was conducted on female and male mice with 4 wild types (WT) and 4 TLR2 KO per group. Tissue samples of the cortex, cerebellum, and hippocampus were isolated by neuroanatomical dissection and subjected to membrane fractionation protocol to obtain synaptic junction enriched fractions. Tryptic in- gel digestion was applied to the collected fraction, and generated peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC- MS/MS). Data analysis revealed significant changes in protein abundance across all isolated brain regions and supported our previous results demonstrating alterations of Np and P-Type ATPases expression in TLR2KO mice. The most remarkable and sex-dependent differences in protein content were observed in the cortex of TLR2KO animals compared to WT, while a lesser extent of proteome disruption was shown in the hippocampus and cerebellum of TLR2KO animals. The protein systems that underwent significant modifications are particularly those implicated in neurotransmission, overall pre-and postsynaptic arrangements, synaptic energy metabolism, axon/dendrite cytoskeleton, and myelin sheath modifications. In conclusion, investigating a synaptic proteome in the TLR2KO mouse model enables a fresh look at the potential new roles of TLR2 in intercellular communication and neuronal connectivity in a sexspecific manner.

Toll-like receptor 2 (TLR2) deficient mice model is widely used for investigation of microglial role in regulating inflammatory response following ischemic brain lesion (1). Additional functions and broader cell-type expression of TLRs have been evidenced in brain tissue which makes TLRs knock- out mice (such as TLR2-KO) a promising model for investigating neuron-microglia interactions, and microglial regulation of plasticity and excitability (2). Plasma membrane Ca2+ ATPases (PMCAs) participate significantly in ion homeostasis maintenance and signal transduction in brain tissue, thus we aimed to examine PMCAs protein expression and catalytic activity across brain regions in TLR2-KO mice. Results acquired by Western blot analysis and spectrophotometric enzyme activity measurements exhibit strikingly different protein expression and enzyme activity in brain tissue samples derived from TLR2-KO when compared with wild-type (WT) mice. Observed abundance of PMCA expression across cortex, hippocampus and cerebellum suggests that ubiquitous isoforms, PMCA 1 and 4 are less affected by TLR2 deficiency than neuron specific isoforms, PMCA 2 and 3. Notably, higher expression of PMCA2 in hippocampus and lower in cerebellum of TLR2-KO coincide with catalytic PMCA activity variations, higher activity is detected in hippocampus and lower in cerebellum of TLR2-KO compared with WT controls. Here collected evidence suggests that TLR2 deficiency is accompanied by a disturbance of neuronal Ca2+ signaling system. Further investigation should clarify in more details a potential impact of TLR2 on establishing proper neuron-microglia interactions and regulation of neurotransmission. [1.] Gorup D, Škokić S, Kriz J, Gajović S. Tlr2 Deficiency is Associated with Enhanced Elements of Neuronal Repair and Caspase 3 Activation Following Brain Ischemia. Sci Rep. 2019 ; 9(1):1–10. [2.] Yamamoto M, Kim M, Imai H, Itakura Y, Ohtsuki G. Microglia- Triggered Plasticity of Intrinsic Excitability Modulates Psychomotor Behaviors in Acute Cerebellar Inflammation. Cell Rep [Internet]. 2019 ; 28(11):2923-2938.e8. Available from: https://doi.org/10.1016/j.celrep.2019.07.078 [3.] Hajieva P, Baeken MW, Moosmann B. The role of Plasma Membrane Calcium ATPases (PMCAs) in neurodegenerative disorders. Neurosci Lett. 2018 ; 663(September 8. Available from: https://doi.org/10.1016/j.neulet.2017.09.033 Funding: Croatian Science Foundation (IP-2016-06- 8638), European Social Fund (ESF), and Centre of Excellence for basic, clinical and translational neuroscience.

Transmembrane proteins, neuroplastin (Np), and Toll-like receptor 2 (TLR2) are implicated in tuning the neuro-architecture. Np isoforms (Np55 and Np65) are related to synaptic plasticity and neuronal ion homeostasis by interactions with plasma membrane Ca2+ ATPases (PMCA). TLR2 is mainly expressed on microglia, but much broader cellular localization of TLR2 has been established, particularly during neurodevelopment. To test the hypothesis that TLR2 deficiency affects membrane dynamics, we analysed membrane protein phenotype in the brains of TLR2 knock-out (KO) mice. Cortical, cerebellar, and hippocampal tissue derived from male TLR2-KO and age-matched control (C) mice (N=36+36) underwent systematic biochemical profiling encompassing: analysis of Np55, Np65, PMCA, Na+/K+-ATPase (NKA) expression by Western blot (WB) ; transcriptional variations of selected genes by qPCR ; synaptic proteome by mass spectrometry (MS) ; activity of NKA and PMCA by spectrophotometry. MS analysis revealed astonishing differences in the synaptic proteome. Increased Np55, Np65, and NKA expression in cortex, cerebellum, and hippocampus was determined by WB in TLR2 vs. C. These findings were confirmed at a transcriptional level using qPCR. WB analysis of 4 PMCA isoforms showed that lack of TLR2 is associated with decreased expression of PMCA2 in cortex and cerebellum and increased in hippocampus. Changes in NKA and PMCA activity were also observed in TLR2 KO vs. C. Multiple-level analysis revealed that TLR2 deficiency leads to altered expression of proteins associated with synaptic plasticity and ion homeostasis. Observed proteomic changes are related to glutamatergic transmission, neuronal cytoskeleton organization, and mitochondrion energy metabolism. Further investigation may clarify previously undescribed roles of TLR2 in neuron-microglia interactions, synaptic connections arrangement, and neurotransmission.

Populations around the world are ageing faster than ever in the past. A constant and already impressive rate in the worldwide increase of life expectancy has led to the fact that the current proportion of the population above 60 years (17%) will double in the next thirty to forty years. In the next 30 years, every third person in the world will fall into the category of a senior citizen. This demographic transition will have an impact on almost all aspects of society and requires a complete and well-defined shift in the paradigm in the medical, social, and technological fields.

Aim of the study was to determine the immunoreactivity pattern and unravel potential changes of two isoforms of synaptic transmembrane glycoprotein neuroplastin (Np55 and Np65) in brains of Toll-like receptor 2 deficient (TLR2D) mice in comparison with wild-type (WT) animals. For the purposes of immunohistochemical analysis by confocal microscopy, mouse brain sections derived from TLR2D and WT mice (n=10) were subjected to immunofluorescence labeling using antibodies against Np55, Np65, TLR2, and specific cell-type markers for neurons and different glial cells. Our preliminary Western blot analysis of cortical, hippocampal and cerebellar membrane fractions revealed changed expression levels of two Np isoforms in TLR2D vs WT mice. Here presented immunohistochemical data indicate higher Np55 and Np65 signal intensity in cortical and hippocampal area of TLR2D vs WT mice, and lower Np55 signal intensity in cerebellum of TLR2D mice, which corresponds to our previous Western blot results. In addition, Np was found to be expressed only in neuronal cells in both TLR2D and WT mice. TLR2D mice are a widely used model for studying neuroinflammatory component of different neuropathological disorders and Np is known to be involved in synaptic organization and affected in neurodegenerative disorders. It may be expected that deficiency of TLR2 has impact on cell-cell communication due to changed molecular signatures of neurons and surrounding glial cells. Having in mind evidenced role of TLR2D in activated microglia, deciphering biochemical phenotype of TLR2D mice could as well contribute to understanding potential involvement of neuroplastin in neuroinflammation.

Toll-like receptors (TLRs) are widely expressed in mammalian brain. Involvement in neurodegeneration and hypoxia has been evidenced for several microglial TLRs. In order to investigate interplay of specific microglial and neuronal transmembrane proteins and its potential role in neurodegeneration, we use TLR2 knock-out (KO ) mice (C57BL/6 TLR2-/-).Our preliminary findings indicate different expression of synaptic transmembrane protein neuroplastin (Np) in brain tissue of TLR2 KO mice in comparison with wild- type littermates (WT).Neuroplastin, a cell- adhesion molecule found in two isoforms (Np65 and Np55) is an essential auxiliary subunit of plasma membrane Ca2+ ATPase (PMCA ). In addition, Np is influenced by glycosphingolipid environment of the membrane, which is also the case for another ion pumping enzyme, Na+/K+-ATPase (NKA). Since Np, PMCA and NKA all seem to favor specific lipid milieu in order to function optimally, and at least some of them are confirmed to interact with one another, we investigated the expression of these proteins in TLR2 KO compared to WT across different brain regions.We isolated membrane fractions from cortical, hippocampal and cerebellar tissue of 5 WT and 5 KO animals, and analyzed the expression of Np, NKA and PMCA by Western blotting. Data revealed increased expression level of Np isoforms and PMCA in cortex and hippocampus in TLR2 KO, and decreased expression of Np55 and PMCA in the cerebellum. NKA exhibited the same expression pattern in TLR2 KO vs WT, and increased expression in all regions analyzed (cortex, hippocampus, cerebellum). Furthermore, we investigated the cortical NKA activity which is lower in TLR2 KO mice.This data together with ongoing immunohistochemistry and transcriptional analysis will enable more detailed neurobiochemical characterization of TLR2 KO model, and give insight into functional implications of differential regional distribution of here analyzed proteins involved in synaptic plasticity and membrane ion transport.

Priručnik daje pregled metoda i pristupa u istraživanju i liječenju bolesti mozga koji se temelje na matičnim stanicama.

The aim of this study was to investigate the expression of two isoforms of synaptic glycoprotein neuroplastin (Np), Np55 and Np65, in the brain cortex and cerebellum of mouse lacking Toll-like receptor 2 (TLR2). Since TLRs are widely expressed in mammalian brain and involved in neurodegeneration and hypoxic stress, TLR2 knock-out (KO) mouse model (B6.129-Tlr2tm1Kir/J) is especially intriguing to analyze Np expression due to following evidence: 1) there is a threefold increase in Np65 in rat forebrain following transient ischemia suggesting a role of Np65 in recovery from ischemic insult ; 2) Np contributes to neuronal energy metabolism by acting as chaperone of monocarboxylate transporter MCT2 ; 3) Np65 KO mice have been found to be more susceptible to ischemic brain lesions. In TLR2 KO mice, attenuated neurodegeneration upon hypoxic stimuli has been described due to reduced microglial activation. To fill in the gaps in biochemical description of this model we investigated expression of Np55 and Np65 isoforms in brain tissue derived from wt and TLR2 KO mice. Membrane fractions were isolated from cortical and cerebellar tissue samples dissected from 22 animals, in which expression of two Np isoforms was determined by Western blotting. Results showed statistically significant increase of both Np55 and Np65 in cortex and decrease of Np55 in cerebellum in male KO vs wt mice. Obtained data are introduction for a larger study which will address potential interplay of Np and TLR2 as well as influence of hypoxia on Np expression in different brain regions.

Spinal root avulsion results in paralysis and sensory loss, and is commonly associated with chronic pain. In addition to the failure of avulsed dorsal root axons to regenerate into the spinal cord, avulsion injury leads to extensive neuroinflammation and degeneration of second-order neurons in the dorsal horn. The ultimate objective in the treatment of this condition is to counteract degeneration of spinal cord neurons and to achieve functionally useful regeneration/reconnection of sensory neurons with spinal cord neurons. Here we compare survival and migration of murine boundary cap neural crest stem cells (bNCSCs) and embryonic stem cells (ESCs)- derived, predifferentiated neuron precursors after their implantation acutely at the junction between avulsed dorsal roots L3-L6 and the spinal cord. Both types of cells survived transplantation, but showed distinctly different modes of migration. Thus, bNCSCs migrated into the spinal cord, expressed glial markers and formed elongated tubes in the peripheral nervous system (PNS) compartment of the avulsed dorsal root transitional zone (DRTZ) area. In contrast, the ESC transplants remained at the site of implantation and differentiated to motor neurons and interneurons. These data show that both stem cell types successfully survived implantation to the acutely injured spinal cord and maintained their differentiation and migration potential. These data suggest that, depending on the source of neural stem cells, they can play different beneficial roles for recovery after dorsal root avulsion.

Introduction: This study was based on the mouse strain B6.Cg-Tg(Thy1-YFP)16Jrs/J which under the control of Thy1 gene promoter expresses yellow fluorescent protein (YFP) in all parts of neurons. In this study, we have determined and compared expression pattern of: 1) Thy1 - YFP positive cells during in vitro differentiation of neural stem cells (NSC), 2) during embryonic development of Thy1 – YFP mouse embryo and 3) after transplantation of Thy1 – YFP cells into the stroke-affected mouse brain. Materials and Methods: Neural stem cells were isolated from the forebrain of 14.5 old mouse embryos and cultured as neurospheres. Immunocytochemistry and RT-PCR were performed after cells differentiation. Embryonic slices were prepared for immunohistochemistry. In third part of this study, PKH26 labeled NSC were transplanted into the stroke-affected mouse brain obtained by MCAO method. Results: THY1 - YFP expression was described during differentiation of NSC and observed in neural progenitors as well as in mature neurons. Neural progenitors were nestin positive while mature cell were not. THY1 – YFP positive cells were positive for mature neuronal markers (MAP2, β3-tubulin and NeuN) during the whole differentiation period, while astrocytes which were GFAP positive were not expressing YFP. During embryonic development THY1 – YFP positive cells were observed from E12.5 and the expression steadily increased to the end of pregnancy. Positive cells were present in both, central and peripheral nervous system. After transplantation, cells survived and incorporated into the stroke-affected brain. Conclusion: Our results showed that mature neurons as well as neuronal progenitor cells do express THY1 – YFP construct in in vitro conditions, during embryonic development and after transplantation into the stroke-affected mouse brain. This suggests that in addition to analyses of neuronal differentiation in B6.Cg- Tg(Thy1-YFP)16Jrs/J mouse, NSCs isolated from this strain can be successfully used in studies where tracing of cells with neuronal fate is needed.

INTRODUCTION: Biocompatibility, safety and risk assessments of superparamagnetic iron oxide nanoparticles (SPIONs) are of the highest priority in testing their potential applications in biomedicine. Improvement of the biological properties of SPIONs may be achieved by different functionalization and surface modifications. AIMS: This study investigated how different surface functionalizations of SPIONs – uncoated, coated with D-mannose or coated with poly-L-lysine - affected biocompatibility. By using murine neural stem cells (NSCs) as important model system for the regenerative medicine we had a goal to reveal the possible mechanism of toxicity of SPIONs on NSCs, mitochondrial membrane potential, cell membrane potential and DNA damage. RESULTS: We observed hyperpolarization of mitochondrial membrane, dissipated cell membrane potential and increased DNA damages in NSCs, irrespective of the surface coating applied for SPION stabilization. Although surface coating should prevent the toxic effects of SPIONs, our results showed that all of the tested SPION types affected the NSCs similarly indicating that mitochondrial homeostasis is their major cellular target. CONCLUSIONS: Despite the claimed biomedical benefits of SPIONs, the refined determination of their effects on various cellular functions presented in this work highlights the need for further safety evaluations. This investigation helps to fill the knowledge gaps on the criteria that should be considered in evaluating the biocompatibility and safety of novel nanoparticles. ACKNOWLEDGMENTS: This study was supported by EU FP7 grant GlowBrain (REGPOT-2012-CT2012-316120), by EU ESF project MladiMozak (030201-0180) and by the Czech Science Foundation (project no. 16-01128J).

Drugo izdanje priručnika daje sažeti pregled doprinosa molekularne biologije u području genetike i epigenetike, genomike, transkriptomike, proteomike, nutrigenomike i farmakogenomike, te prikaz modernih trendova u primjeni najnovijih dostignuća u kliničkim i znanstvenim laboratorijima.

Objective In order to analyze morphologic processes occurring during development of mammalian CNS, several specific mouse strains have been constructed. This study was based on the mouse strain B6.Cg-Tg(Thy1-YFP)16Jrs/J which under the control of Thy1 gene promoter expresses yellow fluorescent protein (YFP) in neurons. In this study, we have determined and compared pattern of Thy1 - YFP expression during embryonic development as well as during differentiation of neural stem cells culture. Material and methods Pregnant females, at different stages of gestation were used for embryo isolation. The embryos were fixed and cryosectioned on 20µm thick slices. Neural stem cells were isolated from the forebrain of 14.5 old murine embryos and cultured as neurospheres. After dissociation, cells were plated on cover slips. The cells were fixed 1, 3, 5 and 7 and prepared to the immunocytochemistry. Results First signs of expression were observed at embryonic day 12.5 in ventral horn of the spinal cord. During development of the embryo in the cranio-caudal direction. In accordance with the process of neurogenesis which primarily occurs in mid-gestation, expression of YFP in neurons after E12.5 became much more pronounced. With embryo maturation, expression became stronger in the brain and by E17.5 expression was observed in the optic nerve and retina. Furthermore, expression of YFP was determined during in vitro NSC differentiation. YFP expression was observed in neural progenitors as well as in mature neurons. YFP positive cells stained positive for Map2 (mature neuronal marker), while astrocytes which were GFAP positive were not expressing YFP. Conclusion Our results showed that, in addition to mature neurons, neuronal progenitor cells express this construct both in in vivo and in vitro conditions. This suggests that in addition to analyses of neuronal differentiation in B6.Cg- Tg(Thy1-YFP)16Jrs/J mouse, NSCs isolated from this strain can be successfully used in studies where tracing of cells with neuronal fate is needed.

Posteljica igra ključnu ulogu u prijenosu hranjivih tvari potrebnih za normalan fetalni razvoj. Za rast masnog tkiva djeteta pritom su najvažniji glukoza i lipidi. Dok je prijenos glukoze kroz posteljicu već odavno prepoznat kao olakšana difuzija ovisna o koncentracijskom gradijentu, sam mehanizam prijenosa lipida je velikim dijelom nepoznat. Istraživanja upućuju na moguću ulogu lipoproteinske lipaze (LPL) koja u metabolizmu lipida ima veliku važnost, a djeluje kao katalizator hidrolize triglicerida kojom se stvaraju slobodne masne kiseline. U kojoj mjeri je ovaj mehanizam doista važan za prijenos lipida kroz posteljicu nije poznato. Odlučili smo stoga napraviti analizu izražaja navedenog gena u terminskim posteljicama zdravih trudnica i trudnice s nasljednom hipertrigliceridemijom, recesivnim poremećajem uzrokovanim mutacijom LPL gena. Western blot analiza monoklonalnim protutijelom anti-LPL (5D2, Abcam) pokazala je jak signal lipoproteinske lipaze u posteljicama zdravih trudnica (n=6) dok je kod trudnice s nasljednom hipertrigliceridemijom signal bio 50% slabiji. Imunohistokemijska analiza istim anti-LPL protutijelom pokazala je najjaču LPL imunoreaktivnost u trofoblastnim stanicama s nešto slabijim signalom u endotelnim stanicama krvnih žila. Osim navedenih analiza na proteinskoj razini, kvantificirali smo i LPL mRNA pomoću RT- PCR koji je pokazao 3-4 puta veću razinu mRNA kod trudnice s nasljednom hipertrigliceridemijom u odnosu na zdrave trudnice što je vjerojatno posljedica pokušaja kompenzacije nedostatka LPL proteina pojačanom mRNA proizvodnjom. Ovim istraživanjem smo jasno dokazali prisutnost LPL mRNA i proteina u trofoblastnim stanicama posteljica zdravih trudnica u terminu. Usmjeravanjem daljnjeg istraživanja na analizu promjene izražaja LPL gena u pojedinim patološkim stanjima poput gestacijskog dijabetesa ili intrauterinog zastoja u rastu, dobit ćemo bolji uvid u mehanizme koji dovode do promjena u ukupnoj količini fetalnog masnog tkiva i abnormalnog fetalnog rasta u navedenim poremećajima.

U radu su predstavljene osnove regenerativne medicine i dat kratak pregled istraživanja iz tog područja koja svoje izvorište i potencijalnu kliničku primjenu imaju u području dentalne medicine.

In order to analyze morphologic processes occurring during development of mammalian nervous system, several specific mouse strains have been constructed. This study was based on the mouse strain B6.Cg-Tg(Thy1-YFP)16Jrs/J which under the control of Thy1 gene promoter expresses yellow fluorescent protein (YFP) in all parts of neurons. This allows to precisely describe development of otherwise less visible parts of nervous cells, including axon and dendrites. In order to use these cells for transplantation, we had to determine pattern of Thy1 - YFP expression during embryonic development. During the first 10 days of embryonic development expression of YFP was not observed. First signs of expression were observed at embryonic day 12.5 (E12.5) in ventral horn of the spinal cord. During development of the embryo in the cranio-caudal direction, a stronger expression of YFP was observed in the cranial part of the spinal cord. Moreover, expression was significantly stronger in the spinal cord than in the brain. In accordance with the process of neurogenesis which primarily occurs in mid-gestation, expression of YFP in neurons after E12.5 became much more pronounced. With embryo maturation, expression became stronger in the brain and by E17.5 expression was observed in the optic nerve and retina. In the newborn mouse P0 expression was observed in the olfactory bulb. Our preliminary results showed that, except neurons, progenitor cells also express this construct. Therefore, stem cells isolated from Thy1 - YFP at E14.5 can be successfully traced after transplantation in the brain and their processes arising during cell differentiation can be recognized using a bright fluorescence of YFP.

In order to analyze morphologic processes occurring during development of mammalian nervous system, several specific mouse strains have been constructed. This study was based on the mouse strain B6.Cg-Tg(Thy1-YFP)16Jrs/J which under the control of Thy1 gene promoter expresses yellow fluorescent protein (YFP) in all parts of neurons. This allows to precisely describing development of otherwise less visible parts of nervous cells, including axon and dendrites. During the first 10 days of embryonic development expression of YFP was not observed. First signs of expression were observed at embryonic day 12.5 (E12.5) in ventral horn of the spinal cord. During development of the embryo in the cranio-caudal direction, a stronger expression of YFP was observed in the cranial part of the spinal cord. Moreover, expression was significantly stronger in the spinal cord than in the brain. In accordance with the process of neurogenesis which primarily occurs in mid-gestation, expression of YFP in nerve cells after E12.5 become much more pronounced. Thus expression of YFP was observed in the brain, especially at the basis of the brain by E13.5. With embryo maturation, expression became stronger in the brain and by E17.5 expression was observed in the optic nerve and retina. In the newborn mouse P0 expression was observed in the olfactory bulb. Analyses of expression of Thy-YFP construct in B6.Cg-Tg(Thy1-YFP)16Jrs/J strain revealed a high rate of development of nervous system cells starting at E12.5 and increasing during later stages of gestation and reaching the first days of postnatal life.

Neuronal nuclear localization of STAM2 protein, a member of the endosome-associated complex ESCRT-0

Stem cell research raises hopes for incurable neurodegenerative diseases. In amyotrophic lateral sclerosis (ALS), affecting the motoneurones of the central nervous system (CNS), stem cell-based therapy aims to replace dying host motoneurones by transplantation of cells in disease-affected regions. Moreover, transplanted stem cells can serve as a source of trophic factors providing neuroprotection, slowing down neuronal degeneration and disease progression. Aim: To determine the profile of seven trophic factors expressed by mesenchymal stem cells (MSC) and neural stem cells (NSC) upon stimulation with CNS protein extracts from SOD1-linked ALS rat model. Methods: Culture of rat MSC, NSC and fibroblasts were incubated with brain and spinal cord extracts from SOD1(G93A) transgenic rats and mRNA expression of seven growth factors was measured by quantitative PCR. Results: MSC, NSC and fibroblasts exhibited different expression patterns. Nerve growth factor and brain-derived neurotropic factor were significantly upregulated in both NSC and MSC cultures upon stimulation with SOD1(G93A) CNS extracts. Fibroblast growth factor 2, insulin-like growth factor and glial-derived neurotropic factor were upregulated in NSC, while the same factors were downregulated in MSC. Vascular endothelial growth factor A upregulation was restricted to MSC and fibroblasts. Surprisingly, SOD1(G93A) spinal cord, but not the brain extract, upregulated brain-derived neurotropic factor in MSC and glial-derived neurotropic factor in NSC. Conclusions: These results suggest that inherent characteristics of different stem cell populations define their healing potential and raise the concept of ALS environment in stem cell transplantation.

Besides its well-known role in cervical carcinoma, HPV is also suggested to be involved in lung cancer development. A number of authors have been investigating the presence of HPV in histological materials. We used routine bronchial aspirates from 84 patients with lung carcinoma for DNA extraction and then performed polymerase chain reaction for high-risk HPV types 16, 18 and 33. The results were compared to those obtained from buccal and eyelid mucosa. Only three patients were positive for HPV in bronchial aspirates: one for HPV 16 type, one for HPV 18 type, and one for HPV 33. Our data indicated the low prevalence of HPV in patients with lung carcinomas in Croatia, therefore it seems unlikely that HPV contributes to the development of lung carcinomas in this region.

The mouse transcription factor Noto is expressed in the notochord and involved in its development. Noto mouse mutants, Noto(tc/tc)(truncate) and Noto(GFP/GFP) (Noto null mutant), exhibit a segmental lack of the notochord in the caudal part of the embryo and subsequent tail truncation in adult animals. In order to address the relationship between the tail bud (the undifferentiated mesenchymal cells in the tip of the embryo tail) and the caudal notochord, Noto(GFP/GFP), a loss- of-function mutant, was analyzed. Taking advantage of Noto(GFP/+) heterozygotes, we could track Noto-GFP-expressing cells from the tail bud over the tail cord to the caudal notochord, and confirm a morphological continuum from the tail bud mesenchyme to the caudal notochord. Loss of Noto affected tail bud morphogenesis: Noto-GFP-expressing cells were scattered in the tail bud mesenchyme. They segregated in the notochord- like structure within the medullary cord instead at the tail cord, which subsequently disappeared. In the tail cord, instead of the notochord, additional lumen of the tail gut was formed. These findings suggest that Noto is involved in both rearrangement and morphogenesis of the tail bud during notochord formation.

Expression analysis of STAM2 (Signal transducing adaptor molecule 2), subunit of ESCRT-0 endosomal trafficking complex in the mouse central nervous system.

Amyotrophic lateral sclerosis (ALS) is a progressive and terminal neuroinflammatory and neurodegenerative disease specifically affecting motoneurons. Death caused by paralysis occurs only 3 to 4 years after the first observed symptom. Although genetic background of 90% of the cases is not defined, uniformity of pathological features in familial superoxide dismutase 1 (SOD1) mutation yielded high interest for this mutation and corresponding animal model. As recent publications suggested that several molecules (cytokines) which are involved in inflammatory process attract neural stem cells, we decided to test whether cells injected in the rat blood stream will be attracted by pathological process within CNS. Moreover, as undifferentiated neural stem cells express only tumor necrosis factor receptor type 2 (TNF-R2), which protects them from induction of apoptosis by TNFα , we wanted to test whether injection of TNFα will increase their migration to CNS. Rational was the well known permeabilization of blood brain barrier by TNFα . After injection of 10 millions of wild type nestin/GFP positive cells, some were found scattered in the brain cortex. More cells were found in both disease affected animals and TNFα treated wild type animals than in untreated wild type animals. This suggests that increased levels of cytokines in the CNS of ALS affected animal might increase chemo - attraction of neural stem cells towards the inflamed neural tissue.

Amyotrophic lateral sclerosis (ALS) is a fatal and still incurable neurodegenerative disease characterized by fast deterioration of motor neurons. Death caused by paralysis occurs only 3 to 4 years after the first symptom is observed. Although majority of the cases are sporadic, familial subtype of ALS characterized by dominant inheritance attracted attention for mutation in superoxide dismutase 1 (SOD1) gene. In order to get insight in the pathological background of ALS, corresponding rat and mouse model encompassing various modifications of SOD1 gene are constructed. Moreover, availability of SOD1 animal models offers possibility to test various therapeutic approaches, including cell therapy and drug screening. In order to test potential of neural stem cell therapy in the rat model of ALS, in vitro differentiation of neural stem cells obtained from embryo cortex of SOD1 mutant rat was analyzed. Observed facts included shift in expression of various differentiation markers and disturbed cell clustering. In order to test potential beneficial effect using blood delivery approach, 10 millions of wild type nestin/GFP positive cells were injected in the rat tail vein. Interestingly, after 3 days, injected cells were found in the cortex and hippocampus of treated animals. Various ALS animal experimental models enable to test innovative therapeutic approaches for this fatal disease. Presence of injected neural stem cells in different regions of ALS affected rat brain after blood stream delivery represents important step further in cell transplantation therapy.

Common pathological features of neurodegenerative diseases are progressive dysfunction and dying of neurons. In amyotrophic lateral sclerosis (ALS), motor neurons are selectively affected, leading to death due to paralysis. The main therapeutic goal in neurodegenerative diseases is to diminish neural dysfunction and to replace non functional cells with the new ones. “ Cell – oriented” treatment strategies include isolation of neural stem cells, their controlled differentiation, and cellular injections targeting the affected region. Expected effects of injected cells result from the combination of cell replacement and re- establishment of natural cellular environment. Here we summarize the current state of isolation and differentiation of neural stem cells, and we emphasize the region of the tail bud development as natural region where neuroepithelium is derived from undifferentiated mesenchymal cells. The possibility to obtain cells from autologous mesenchyme capable to integrate in affected regions represents a major challenge whose achievement should circumvent the pitfall of the immune reaction against transplanted cells. In this work, we present the current knowledge and clues needed to generate neural stem cells and we illustrate their potentials. Moreover, we present our own results: intravenously injected neural stem cells in symptomatic ALS rats crossed blood brain barrier, migrated to the motor cortex, and continued to differentiate. Thus, the use of neural stem cells in rodent models of amyotrophic lateral sclerosis represents a paradigm for all other neurodegenerative diseases.

Analysis of expression of Stam2 gene using a gene trap derived transgenic mouse model

Expression analysis of Stam2 gene in the brain using a gene trap derived transgenic mouse model

Amyotrophic lateral sclerosis (ALS) is a progressive and still incurable neurodegenerative disease characterized by dying of motor neurons, which leads to fatal paralysis and death. Facts that majority of affected individuals are coming from economically active middle-aged population and that they die paralyzed only 3 to 4 years after the first observed symptoms attract attention of the wide biomedical community. The last decade was marked by amazing progress in the field of the neural stem cells (NSC) science. Developed techniques of isolation and differentiation, including obtaining of stem cells from adult tissue opened a wide range of possibilities to test completely new therapeutic approaches in neurodegenerative diseases. Herewith, we report our work with NSC and ALS animal model. In order to get insight in the possible therapeutic application of NSC in a rat model of ALS, specific protocol was developed. It included use of rat experimental model based on superoxide dismutase 1 (SOD1) mutation, characterized by high uniformity of pathological features and high similarity to human disease. NSC from the cortex of 16 days old wild type rat embryos were successfully isolated, neurospheres were cultivated, and high number of undifferentiated and viable cells were obtained. Injection of GFP marked NSC within blood stream of affected animals resulted with presence of the cells within disease affected motor cortex. After 3 days, cells started to differentiate and both neural (Map2 positive) and astrocytic (GFAP positive) markers were recognized. Although approximately 35% of Map2+ cells died by apoptosis, probably caused by cell integration failure within host tissue, remaining Map2+ and majority of GFAP+ cells continue to be present in the motor cortex. Preliminary results showing the presence of synaptic marker synaptotagmin suggest that transplanted cells can survive and integrate within disease affected region. The presence of injected NSC in motor cortex of ALS affected rat brain represent an important step further in cell transplantation therapy of this neurodegenerative disease.

OBJECTIVE: To get insight in the possible therapeutic application of neural stem cells in a rat model of ALS. BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive and terminal neurodegenerative disease defined by fast deterioration of motoneurons. Although represented with relatively low prevalence of 0.1 ‰ , a fact that majority of affected individuals die paralyzed only 3 to 4 years after the first observed symptoms attracts high attention of the wide biomedical community. The most often used experimental model is superoxide dismutase 1 (SOD1) rat mutant, characterized by high uniformity of pathological features and high similarity to human disease. RESULTS: In order to get insight in the possible therapeutic application of neural stem cells in a rat model of ALS, specific protocol was developed. It was based on isolation of neural stem cells from the cortex of 16 days old wild type rat embryos. Cultivation of small to medium sized neurospheres in neural stem specific medium enabled to obtain high number of undifferentiated and viable cell, as it was required that injected cells retain capability to migrate to the region affected by neurodegeneration. Moreover, as ALS is not defined by strict regional localization, it was of high interest to test whether blood delivery could be efficient in reaching different regions of motocortex and spinal cord. To test this approach, 10 millions of cells were injected in SOD1 mutant rat tail vein. After 3 days, injected cells were found scattered in the cortex and hippocampus. CONCLUSION: Presence of injected neural stem cells in different regions of ALS affected rat brain represents important step further in cell transplantation therapy of this neurodegenerative disease.

Amyotrophic lateral sclerosis (ALS) is a progressive and terminal neurodegenerative disease specifically affecting motoneurons. Death caused by paralysis occurs only 3 to 4 years after the first observed symptom. Although genetic background of 90% of the cases is not defined, uniformity of pathological features in familial superoxide dismutase 1 (SOD1) mutation yielded high interest for this mutation and corresponding animal model. In order to get insight in the cellular mechanisms involved in pathogenesis of amyotrophic lateral sclerosis, in vitro differentiation of neural stem cells obtained from embryo cortex of SOD1 mutant rat were compared to the wild type control. Moreover, their therapeutic potential using blood delivery was tested. In vitro differentiation of SOD1 cells revealed differences in comparison to their wild type counterparts. While in wild type cell cultures presence of nestin marked early phase in the neural stem cells development, 100% of cells in SOD1 cultures with advanced neuron - like morphology were simultaneously expressing nestin and neuron specific MAP2. Moreover, major differences in cell clustering patterns were observed. In order to test whether in vitro cultured cells can be delivered through circulation to the brain of symptomatic SOD1 mutant rat, 10 millions of wild type nestin/GFP positive cells were injected in the rat tail vein. After 3 days, cells were found in the cortex and hippocampus of treated animals. As pathological process in amyotrophic lateral sclerosis affects scattered regions of the brain and the spinal cord, adapted therapeutic blood delivery based approach was applied. We believe that presence of injected neural stem cells in different regions of ALS affected rat brain represents important step further in cell transplantation therapy of this neurodegenerative disease.

The fact that embryo tail morphogenesis and elongation occur long after gastrulation is completed resulted in controversy about the mechanisms of the tail bud differentiation. The formation of embryo tail structures, in particular the neural tube, the notochord, and the tail gut, could be outcome of the interactions among gastrulation - predetermined germ layers, or alternatively, origin of the tail structures could be traced to the mesenchymal stem cells of the tail bud. To analyze genetic markers of the tail bud development in the mouse embryo and to get insight in the mechanisms of the tail bud differentiation, mouse mutant embryos with loss of function of Noto gene were compared to their wild type counterparts, and to the Noto spontaneous mutant referred as truncate. In addition, in frame- inserted GFP within the Noto gene served as a marker of developing notochord In the wild type 11.5-days old embryos, the tail bud mesenchyme was continuous to the two cords ; the medullary cord located dorsally, and the tail cord located centrally within the embryo tail. The medullary cord extended into the neural tube by the process of secondary neurulation, i.e. rearrangement of mesenchymal cells during formation of the neural tube. The tail cord extended into both notochord and the tail gut. To test whether morphological continuum of the notochord and the tail bud cells was related to its origin from the tail bud mesenchyme, the expression of Noto was analysed. Indeed, the Noto expression was present specifically in the tail notochord and extended well into the tail bud. In contrast to the narrow notochord, the expression spread in the wider region of tail bud cells. In order to get insight in Noto function during tail bud development, the homozygous Noto mutants were analysed. Instead of the tail notochord, which was missing in the long segments of the mutant tails. the notochord-like cells arose from the medullary cord together with cells involved in the secondary neurulation. Instead of the notochord, part of the tail cord gave rise to the additional tail gut. To clarify which of the observed malformed structures expressed the notochord markers, Noto expression was analyzed in the homozygous Noto mutants. Noto expression was still present within undifferentiated tail bud mesenchyme and subsequently confined to the notochord-like cells within the medullary cord. In conclusion, the presented data suggest potential of the tail bud cells to generate notochord. Formation of the notochord from the medullary cord, and the additional tail gut from the tail cord support hypothesis of the tail bud cells pluripotentcy.

Razvoj kaudalnog dijela zametka kralješnjaka odvija se nakon završetka gastrulacije i nestanka primitivne pruge sekundarnim mehanizmom razvoja, tijekom kojeg strukture osnove repa nastaju od nediferenciranih stanica repnog pupoljka. Kaudalni dio zametka poremećen je u mišjih mutanata splotch i truncate. U splotch miševa mutiran je gen Pax3, što ima za posljedicu otvorenu neuralnu cijev (spina bifida) i poremećaj migracije stanica neuralnog grebena. U truncate miševa mutiran je gen Noto, gdje je poremećen razvoj notokorda. Hipoteza ovog istraživanja bila je da je fenotip mutiranih miševa splotch i truncate posljedica nepravilnog razvoja repnog pupoljka, te poremećenog sekundarnog mehanizma razvoja. Cilj rada bio je usporediti razvoj kaudalnog dijela zametka visoko srođenog soja C57Bl/6 s poremećenim razvojem mutanata splotch i truncate. Građa mišjih zametaka starih od 9 do 12,5 dana proučavana je svjetlosnom mikroskopijom serijskih polutankih rezova obojanih toluidinskim modrilom. U zamecima C57Bl/6 miševa sekundarni mehanizam razvoja obuhvaća postupnu diferencijaciju stanica repnog pupoljka, koje se dijele na moždinski tračak od kojeg nastaje sekundarna neuralna cijev, i repni tračak od kojeg nastaje notokord i repno crijevo. Poremećena neurulacija u splotch zametaka nije praćena poremećajem građe repnog pupoljka, osim u splotch zametaka starih 12,5 dana, gdje dolazi do sveobuhvatnog poremećaja razvoja osnove repa kaudalno od spine bifide. Spina bifida u splotch zametaka nastaje na prijelazu iz primarne u sekundarnu neuralnu cijev. U truncate zametaka djelomično nedostaje notokord. Diferencijacija repnog pupoljka je poremećena, te se notokord umjesto od repnog tračka odvaja od ventralne strane sekundarne neuralne cijevi. Dorzalni dio repnog tračka umjesto notokorda oblikuje dodatni lumen repnog crijeva. Nedostatak notokorda u osnovi repa povezan je s poremećajem razvoja susjednih struktura, neuralne cijevi i somita. Opisana morfogenetska zbivanja doprinose razumijevanju razvojnih poremećaja u čovjeka - spine bifide, sindroma kaudalne disgeneze i Waardenburgovog sindroma.

The morphological analysis represents the basic method used to determine the phenotype changes of the genetically modified mouse. In this way the observed phenotype changes are correlated to the genetic modification and enable the insight in the function of the investigated gene. The challenge of the morphological analysis increases with the increased complexity of the investigated tissues and organs. Therefore the most complex organ to be studied is the brain. In order to compare the brain neuroanatomy of the mutant and wild type mice, the brain could be studied at the gross-morphology level as a whole, at the light microscope level on histological sections, or at the ultrastructural level by electron microscopy (1). In order to compare the brain morphology at the ultrastructural level of the mutant and the wild type mouse, we used the mouse mutant Stam2Gt1Gaj, where due to the gene trap modification the function of Stam2 was hindered. Due to the brain size and the nature of electron microscopy analyisis only two brain regions were selected to be viewed by electron microscope, CA2 region of the hippocampus and the adjacent cerebral cortex. In order to compare the equivalent regions of the brain in the mutated and wild type mice the regions of interest were determined on vibratome sections, and the selected pieces of tissues were processed for electron microscopy. Adult, 3 months old mice were anesthetized with 1% avertin and perfusion fixation was performed. 0.1 M phosphate buffer was injected within the left heart ventricle in order to rinse the blood from the circulatory system. Afterwards, the animals were perfused with 12.5% glutaraldehyde, which resulted with initial fixation of the brain. Skull was opened with sharp scissors and the brain was removed and immersed in 6% glutaraldehyde for 1 hour. The brain was afterwards cut in 200 µ m thick sagital sections using vibratome. After determination of the brain regions of interest, the tissue was washed in 0.1 M phosphate buffer and postfixed in 1% osmium tetroxide for 2 hours. Subsequent to contrasting in uranyl acetate over night, the tissue was dehydrated in ethanol and embedded in Durcopan (Fluka). 1 µ m thick semithin sections were cut using the ultramicrotome and examined by light microscopy (figure 1). The selected and comparable regions of the semithin sections were trimmed and 70nm thick ultrathin sections were made, contrasted with lead citrate and uranyl acetate and examined by electron microscopy on transmission electron microscope Zeiss902A (2) (figure 2). The described procedure enabled the comparison of the brain ultrastructure of mice with different genetic background. The comparison was complemented with the photographing of the serial frames of the selected regions in order to increase the size of area for analysis and acquire the necessary data to be used in image analysis applications. Nevertheless, the variability of the tissue examined should be add to the variability among the mice belonging to the same genotype. The presented example highlights the difficulties of morphology analyisis of the genetically modified mice.

Endosomes are involved in the CNS in a variety of actions including formation of synaptic vesicles, axonal transport and receptor sorting. To elucidate function of Stam2 in the CNS, gene trap approach was applied. The expression pattern analysis showed that Stam2 was expressed in developing neural tube and heart. In addition, alternatively spliced variant of Stam2 was found.

STAM2 (Signal transducing adaptor molecule 2) is a signaling adaptor presumed to act in the intracellular cargo sorting for degradation at the level of the late endosome (1). Although its function was already assessed at the cellular level in permanent cell cultures, its function in mammalian organism could be revealed only through genetic modification at the level of the whole animal. In order to characterize Stam2 function in mouse, the gene trap method was applied. It comprises the genetic modification of embryonic stem cells with a non-homologous vector containing a splice acceptor and fused lacZ and neoR genes without their own promoters. In case the vector was inserted in an endogenous gene, the splice acceptor would be recognized and lacZ and neoR genes would be driven by the promoter of the endogenous gene. This enabled to screen the genetically modified embryonic stem cells and the corresponding mouse lines for lacZ activity, which mirrored the expression of the endogenous gene. In addition the vector insertion disturbed the proper protein translation 3’ to the gene trap vector. After the selection process, the mouse line carrying the gene trap mutation in Stam2 gene was chosen for further characterization. The molecular analysis defined the exact place of the insertion and its consequences on the mRNA and the protein level. This showed that the gene trap vector was between exon 2 and 3, and the STAM2 protein production in the homozygous mice was highly diminished (2). Stam2 expression pattern was assessed by monitoring lacZ activity in the investigated tissues. lacZ gives rise to beta-galactosidase, which changes the color of its substrate X-gal in blue. Stam2 expression started in 8.5-day embryos in the hind gut. In 9.5-day embryos it was present in the heart, while in 10.5-day embryos appeared in the ventral part of the neural tube (Figure 1). The expression in the heart persisted during embryo development and continued in the adults. The expression in the neural tube was continuous to the expression in the brain, in particular from 16.5-day embryos in the brain cortex, and from 18.5-day embryos in the hippocampus. In addition to the brain and the heart, Stam2 was expressed as well in the testis and developing adrenal and pituitary glands. In order to assess Stam2 function the phenotype of the homozygous carriers of gene trap mutation was compared with the wild type mice. The homozygotes were alive, fertile and did not show any major abnormalities. The functional studies of mouse behavior were combined with detailed morphological analysis of embryos and adult organs and tissues. The morphological studies involved the classical histology using the hematoxylin-eosin staining, the semithin sections stained by toluidine blue, immunohistochemistry of the selected markers, and finally the ultrastructural analysis using electron microscope (Figure 2). These studies are expected to reveal the fine differences in phenotypes of the wild type and the mutant mice, which would then reveal the Stam2 function in mouse, in particular in the mouse brain.

Development of the caudal part of the mammalian embryo includes differentiation of mesenchymal cells, named the tail bud, in the posterior end of the tail. The tail bud is continuous with the neural tube, notochord and the tail gut, which led to the conclusion that caudal structures of the embryo emerge from the tail bud (1). As experimental manipulations within mammalian embryos are limited by intrauterine location and small dimensions of the embryo, mouse mutants are invaluable tool for investigation of developmental mechanisms. Development of the caudal part of the truncate and splotch mouse mutants, where tail development is affected, was analyzed. Splotch is a spontaneous Pax3 gene mutant, while truncate is a spontaneous Noto gene mutant. Splotch homozygotes die during embryo development showing neural tube, neural crest and somite malformations (2). Corresponding mutation in human causes Waardenburg syndrome characterized by defects of head and hand development and localized absence of pigmentation. Truncate homozygotes have truncated tail, due to the partially absent notochord (3). Splotch and truncate homozygous embryos and wild type control C57BL/6 embryos aged from 9.0 to 12.5 days were isolated, fixed in a mixture of 1% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer and postfixed in 1% osmium tetroxide. They were embedded in Durcopan (Fluka) and serial semithin sections, perpendicular to the longitudinal tail axis, were obtained, stained with toluidine blue and examined by light microscopy. Splotch embryos from 9.0 to 12.5 days showed open neural tube in the embryo tail. In the 9.0 to 11.5 days old embryos development of other axial structures was not influenced by abnormal neural tube. All axial structures were normal and continuous with the tail bud. In contrast, splotch 12.5 days old embryos showed open neural tube accompanied with disturbed development of other axial structures and disturbed morphology of the tail bud. It is suggested that a longer presence of open neural tube influenced development of all structures caudal from the defect. Truncate homozygous embryos exhibited partially disturbed development of the notochord, which was not continuous to the tail bud, but to the ventral neuroepithelium. In addition, discontinuous or, even doubled notochord was found. Lack of the notochord in the caudal part of the tail was accompanied with disturbed development of the neural tube and the somites. Neural tube was smaller in diameter, it lacked lumen and the massive cell death was found in the neuroepithelium. In the parts of the tail without the notochord, the somites were connected in the midline of the tail. It is suggested that disturbed development of the tail bud is a major cause of truncate phenotype. This investigation confirmed continuity of the tail bud and the tail axial structures in the mouse. The observed malformations indicate that the normal sequence of tail development requires the coordinated interactions among the tail axial structures, which can be related to pathogenesis of spina bifida in humans.