Coloration signals the ability to cope with elevated stress hormones: effects of corticosterone on growth of barn owls are associated with melanism.

Stressful situations during development can shape the phenotype for life by provoking a trade-off between development and survival. Stress hormones, mainly glucocorticoids, play an important orchestrating role in this trade-off. Hence, how stress sensitive an animal is critically determines the phenotype and ultimately fitness. In several species, darker eumelanic individuals are less sensitive to stressful conditions than less eumelanic conspecifics, which may be due to the pleiotropic effects of genes affecting both coloration and physiological traits. We experimentally tested whether the degree of melanin-based coloration is associated with the sensitivity to an endocrine response to stressful situations in the barn owl. We artificially administered the mediator of a hormonal stress response, corticosterone, to nestlings to examine the prediction that corticosterone-induced reduction in growth rate is more pronounced in light eumelanic nestlings than in darker nest mates. To examine whether such an effect may be genetically determined, we swapped hatchlings between randomly chosen pairs of nests. We first showed that corticosterone affects growth and, thus, shapes the phenotype. Second, we found that under corticosterone administration, nestlings with large black spots grew better than nestlings with small black spots. As in the barn owl the expression of eumelanin-based coloration is heritable and not sensitive to environmental conditions, it is therefore a reliable, genetically based sign of the ability to cope with an increase in blood corticosterone level.

Superfamily of steroid nuclear receptors: positive and negative regulators of gene expression.

The nuclear hormone receptor superfamily is characterized by an impressive functional diversity of its members despite a remarkable overall structural unity. A variety of ligands bind specifically to them and these receptors control gene networks that have profound effects on growth, development, and homeostasis. The ligand-receptor complexes recognize transcriptional enhancer DNA sequences, the hormone response elements, resulting in induction or repression of gene activity. The similarity between all these hormone response enhancer elements, as well as between the receptors themselves, indicates a conserved general strategy for the hormonal control of transcription by steroids. The activated receptors bind to responsive promoters and most likely mediate the assembly of stage- and tissue-specific transcription factor complexes that stimulate or inhibit gene expression.

Development of nuclear DNA probes for the typing of Trypanososma cruzi

We have developed and tested a new way of typing Trypanosoma cruzi, mamely the use of cloned nuclear DNA fragments as genetic markers. Restriction fragment length polymorphisms were verified on Soutern blots hybridized to random probes. Fragment patterns were analyzed and dendrograms constructed. Our results on well characterized laboratory strains correlate well to published isoenzyme studies. Some of the probes were also hybridized to chromosomes separated by pulse field gel electrophoresis a higher degree of heterogeneity was observed at this level.

Nuclear Factor I genomic binding associates with chromatin boundaries.

BACKGROUND: The Nuclear Factor I (NFI) family of DNA binding proteins (also called CCAAT box transcription factors or CTF) is involved in both DNA replication and gene expression regulation. Using chromatin immuno-precipitation and high throughput sequencing (ChIP-Seq), we performed a genome-wide mapping of NFI DNA binding sites in primary mouse embryonic fibroblasts. RESULTS: We found that in vivo and in vitro NFI DNA binding specificities are indistinguishable, as in vivo ChIP-Seq NFI binding sites matched predictions based on previously established position weight matrix models of its in vitro binding specificity. Combining ChIP-Seq with mRNA profiling data, we found that NFI preferentially associates with highly expressed genes that it up-regulates, while binding sites were under-represented at expressed but unregulated genes. Genomic binding also correlated with markers of transcribed genes such as histone modifications H3K4me3 and H3K36me3, even outside of annotated transcribed loci, implying NFI in the control of the deposition of these modifications. Positional correlation between + and - strand ChIP-Seq tags revealed that, in contrast to other transcription factors, NFI associates with a nucleosomal length of cleavage-resistant DNA, suggesting an interaction with positioned nucleosomes. In addition, NFI binding prominently occurred at boundaries displaying discontinuities in histone modifications specific of expressed and silent chromatin, such as loci submitted to parental allele-specific imprinted expression. CONCLUSIONS: Our data thus suggest that NFI nucleosomal interaction may contribute to the partitioning of distinct chromatin domains and to epigenetic gene expression regulation.NFI ChIP-Seq and input control DNA data were deposited at Gene Expression Omnibus (GEO) repository under accession number GSE15844. Gene expression microarray data for mouse embryonic fibroblasts are on GEO accession number GSE15871.

Biogeographic origin and radiation of the Old World crocidurine shrews (Mammalia: Soricidae) inferred from mitochondrial and nuclear genes.

The crocidurine shrews include the most speciose genus of mammals, Crocidura. The origin and evolution of their radiation is, however, poorly understood because of very scant fossil records and a rather conservative external morphology between species. Here, we use an alignment of 3560 base pairs of mitochondrial and nuclear DNA to generate a phylogenetic hypothesis for the evolution of Old World shrews of the subfamily Crocidurinae. These molecular data confirm the monophyly of the speciose African and Eurasian Crocidura, which also includes the fossorial, monotypic genus Diplomesodon. The phylogenetic reconstructions give further credit to a paraphyletic position of Suncus shrews, which are placed into at least two independent clades (one in Africa and sister to Sylvisorex and one in Eurasia), at the base of the Crocidura radiation. Therefore, we recommend restricting the genus Suncus to the Palaearctic and Oriental taxa, and to consider all the African Suncus as Sylvisorex. Using molecular dating and biogeographic reconstruction analyses, we suggest a Palaearctic-Oriental origin for Crocidura dating back to the Upper Miocene (6.8 million years ago) and several subsequent colonisations of the Afrotropical region by independent lineages of Crocidura.

Design and analysis of ChIP-Seq experiments for nuclear factor I DNA-binding proteins

SUMMARY : Eukaryotic DNA interacts with the nuclear proteins using non-covalent ionic interactions. Proteins can recognize specific nucleotide sequences based on the sterical interactions with the DNA and these specific protein-DNA interactions are the basis for many nuclear processes, e.g. gene transcription, chromosomal replication, and recombination. New technology termed ChIP-Seq has been recently developed for the analysis of protein-DNA interactions on a whole genome scale and it is based on immunoprecipitation of chromatin and high-throughput DNA sequencing procedure. ChIP-Seq is a novel technique with a great potential to replace older techniques for mapping of protein-DNA interactions. In this thesis, we bring some new insights into the ChIP-Seq data analysis. First, we point out to some common and so far unknown artifacts of the method. Sequence tag distribution in the genome does not follow uniform distribution and we have found extreme hot-spots of tag accumulation over specific loci in the human and mouse genomes. These artifactual sequence tags accumulations will create false peaks in every ChIP-Seq dataset and we propose different filtering methods to reduce the number of false positives. Next, we propose random sampling as a powerful analytical tool in the ChIP-Seq data analysis that could be used to infer biological knowledge from the massive ChIP-Seq datasets. We created unbiased random sampling algorithm and we used this methodology to reveal some of the important biological properties of Nuclear Factor I DNA binding proteins. Finally, by analyzing the ChIP-Seq data in detail, we revealed that Nuclear Factor I transcription factors mainly act as activators of transcription, and that they are associated with specific chromatin modifications that are markers of open chromatin. We speculate that NFI factors only interact with the DNA wrapped around the nucleosome. We also found multiple loci that indicate possible chromatin barrier activity of NFI proteins, which could suggest the use of NFI binding sequences as chromatin insulators in biotechnology applications. RESUME : L'ADN des eucaryotes interagit avec les protéines nucléaires par des interactions noncovalentes ioniques. Les protéines peuvent reconnaître les séquences nucléotidiques spécifiques basées sur l'interaction stérique avec l'ADN, et des interactions spécifiques contrôlent de nombreux processus nucléaire, p.ex. transcription du gène, la réplication chromosomique, et la recombinaison. Une nouvelle technologie appelée ChIP-Seq a été récemment développée pour l'analyse des interactions protéine-ADN à l'échelle du génome entier et cette approche est basée sur l'immuno-précipitation de la chromatine et sur la procédure de séquençage de l'ADN à haut débit. La nouvelle approche ChIP-Seq a donc un fort potentiel pour remplacer les anciennes techniques de cartographie des interactions protéine-ADN. Dans cette thèse, nous apportons de nouvelles perspectives dans l'analyse des données ChIP-Seq. Tout d'abord, nous avons identifié des artefacts très communs associés à cette méthode qui étaient jusqu'à présent insoupçonnés. La distribution des séquences dans le génome ne suit pas une distribution uniforme et nous avons constaté des positions extrêmes d'accumulation de séquence à des régions spécifiques, des génomes humains et de la souris. Ces accumulations des séquences artéfactuelles créera de faux pics dans toutes les données ChIP-Seq, et nous proposons différentes méthodes de filtrage pour réduire le nombre de faux positifs. Ensuite, nous proposons un nouvel échantillonnage aléatoire comme un outil puissant d'analyse des données ChIP-Seq, ce qui pourraient augmenter l'acquisition de connaissances biologiques à partir des données ChIP-Seq. Nous avons créé un algorithme d'échantillonnage aléatoire et nous avons utilisé cette méthode pour révéler certaines des propriétés biologiques importantes de protéines liant à l'ADN nommés Facteur Nucléaire I (NFI). Enfin, en analysant en détail les données de ChIP-Seq pour la famille de facteurs de transcription nommés Facteur Nucléaire I, nous avons révélé que ces protéines agissent principalement comme des activateurs de transcription, et qu'elles sont associées à des modifications de la chromatine spécifiques qui sont des marqueurs de la chromatine ouverte. Nous pensons que lés facteurs NFI interagir uniquement avec l'ADN enroulé autour du nucléosome. Nous avons également constaté plusieurs régions génomiques qui indiquent une éventuelle activité de barrière chromatinienne des protéines NFI, ce qui pourrait suggérer l'utilisation de séquences de liaison NFI comme séquences isolatrices dans des applications de la biotechnologie.

Mini-review: Specificity and expression of CIITA, the master regulator of MHC class II genes.

The class II transactivator (CIITA) has been referred to as the "master control factor" for the expression of MHC class II (MHCII) genes. As our knowledge on the specificity and function of CIITA grows, it is becoming increasingly evident that this sobriquet is entirely justified. First, despite extensive investigations, the major target genes of CIITA remain those implicated in the presentation of antigenic peptides by MHCII molecules. Although other putative target genes have been reported, the contribution of CIITA to their expression remains indirect, controversial or comparatively minor relative to its decisive role as a regulator of MHCII and related genes. Second, the most important parameter dictating MHCII expression is by far the expression pattern of the gene encoding CIITA (MHC2TA). The vast majority of signals that activate or repress MHCII expression under physiological and pathological situations converge on one or more of the three alternative promoters that drive transcription of the MHC2TA gene. In short, with respect to its specificity and its exquisitely controlled pattern of expression, CIITA is by a long stretch the single most important transcription factor for the regulation of genes required for MHCII-restricted antigen-presentation.

Looming signals reveal synergistic principles of multisensory integration.

Multisensory interactions are a fundamental feature of brain organization. Principles governing multisensory processing have been established by varying stimulus location, timing and efficacy independently. Determining whether and how such principles operate when stimuli vary dynamically in their perceived distance (as when looming/receding) provides an assay for synergy among the above principles and also means for linking multisensory interactions between rudimentary stimuli with higher-order signals used for communication and motor planning. Human participants indicated movement of looming or receding versus static stimuli that were visual, auditory, or multisensory combinations while 160-channel EEG was recorded. Multivariate EEG analyses and distributed source estimations were performed. Nonlinear interactions between looming signals were observed at early poststimulus latencies (∼75 ms) in analyses of voltage waveforms, global field power, and source estimations. These looming-specific interactions positively correlated with reaction time facilitation, providing direct links between neural and performance metrics of multisensory integration. Statistical analyses of source estimations identified looming-specific interactions within the right claustrum/insula extending inferiorly into the amygdala and also within the bilateral cuneus extending into the inferior and lateral occipital cortices. Multisensory effects common to all conditions, regardless of perceived distance and congruity, followed (∼115 ms) and manifested as faster transition between temporally stable brain networks (vs summed responses to unisensory conditions). We demonstrate the early-latency, synergistic interplay between existing principles of multisensory interactions. Such findings change the manner in which to model multisensory interactions at neural and behavioral/perceptual levels. We also provide neurophysiologic backing for the notion that looming signals receive preferential treatment during perception.

Cell-specific localization of monocarboxylate transporters, MCT1 and MCT2, in the adult mouse brain revealed by double immunohistochemical labeling and confocal microscopy.

Recent evidence suggests that lactate could be a preferential energy substrate transferred from astrocytes to neurons. This would imply the presence of specific transporters for lactate on both cell types. We have investigated the immunohistochemical localization of two monocarboxylate transporters, MCT1 and MCT2, in the adult mouse brain. Using specific antibodies raised against MCT1 and MCT2, we found strong immunoreactivity for each transporter in glia limitans, ependymocytes and several microvessel-like elements. In addition, small processes distributed throughout the cerebral parenchyma were immunolabeled for monocarboxylate transporters. Double immunofluorescent labeling and confocal microscopy examination of these small processes revealed no co-localization between glial fibrillary acidic protein and monocarboxylate transporters, although many glial fibrillary acidic protein-positive processes were often in close apposition to elements labeled for monocarboxylate transporters. In contrast, several elements expressing the S100beta protein, another astrocytic marker found to be located in distinct parts of the same cell when compared with glial fibrillary acidic protein, were also strongly immunoreactive for MCT1, suggesting expression of this transporter by astrocytes. In contrast, MCT2 was expressed in a small subset of microtubule-associated protein-2-positive elements, indicating a neuronal localization. In conclusion, these observations are consistent with the possibility that lactate, produced and released by astrocytes (via MCT1), could be taken up (via MCT2) and used by neurons as an energy substrate.

The role of Pten in the Hematopoietic System and the Hematopoietic Stem Cells

Résumé Identification, localisation et activation des cellules souches hématopoiétiques dormantes in vivo Les cellules souches somatiques sont présentes dans la majorité des tissus régénératifs comme la peau, l'épithélium intestinal et le système hématopoiétique. A partir d'une seule cellule, elles ont les capacités de produire d'autres cellules souches du même type (auto-renouvellement) et d'engendrer un ensemble défini de cellules progénitrices différenciées qui vont maintenir ou réparer leur tissu hôte. Les cellules souches adultes les mieux caractérisées sont les cellules souches hématopoiétiques (HSC), localisées dans la moelle osseuse. Un des buts de mon travail de doctorat était de caractériser plus en profondeur la localisation des HSCs endogènes in vivo. Pour ce faire, la technique "label retaining assay", se basant sur la division peu fréquentes et sur la dormance des cellules souches, a été utilisée. Après un marquage des souris avec du BrdU (analogue à l'ADN) suivi d'une longue période sans BrdU, les cellules ayant incorporés le marquage ("label retaining cells" LCRs) ont pu être identifiées dans la moelle osseuse. Ces cellules LCRs étaient enrichies 300 fois en cellules de phenotype HSC et, en utilisant de la cytofluorométrie, il a pu être montré qu'environ 15% de toutes les HSCs d'une souris restent dormantes durant plusieures semaines. Ces HSCs dormantes à long terme ne sont probablement pas impliquées dans la maintenance de 'hématopoièse. Par contre, on assiste à l'activation rapide de ces HSCs dormantes lors d'une blessure, comme une ablation myéloide. Elles re-entrent alors en cycle cellulaire et sont essentielles pour une génération rapide des cellules progénitrices et matures qui vont remplacer les cellules perdues. De plus, la détection des LCRs, combinée avec l'utilisation du marqueur de HSCs c-kit, peut être utilisée pour la localisation des HSCs dormantes présentes dans la paroi endostéale de la cavité osseuse. De manière surprenante, les LCRs c-kit+ ont surtout étés trouvées isolées en cellule unique, suggérant que le micro-environement spécifique entourant et maintenant les HSCs, appelé niche, pourrait être très réduit et abriter une seule HSC par niche. Rôles complexes du gène supresseur de tumeur Pten dans le système hématopoiétique La phosphatase PTEN disparaît dans certains cancers héréditaires ou sporadiques humains, comme les gliomes, les cancers de l'utérus ou du sein. Pten inhibe la voie de signalisation de la PI3-kinase et joue un rôle clé dans l'apoptose, la croissance, la prolifération et la migration cellulaire. Notre but était d'étudier le rôle de Pten dans les HSC normale et durant la formation de leucémies. Pour ce faire, nous avons généré un modèle murin dans lequel le gène Pten peut être supprimé dans les cellules hématopoiétiques, incluant les HSCs. Ceci a été possible en croissant l'allèle conditionnelle ptenflox soit avec le transgène MxCre inductible par l'interféron α soit avec le transgène Scl-CreERt inductible par le tamoxifen. Ceci permet la conversion de l'allèle ptenflox en l'allèle nul PtenΔ dans les HSCs et les autres types cellulaires hématopoiétiques. Les souris mutantes Pten développent une splénomégalie massive causée par une expansion dramatiques de toutes les cellules myéloides. De manière interessante, alors que le nombre de HSCs dans la moelle osseuse diminue progressivement, le nombre des HSCs dans la rate augmente de manière proportionnelle. Etrangement, les analyses de cycle cellulaire ont montrés que Pten n'avait que peu ou pas d'effet sur la dormance des HSCs ou sur leur autorenouvellement. En revanche, une augmentation massive du niveau de la cytokine de mobilisation G-CSF a été détéctée dans le serum sanguin, suggérant que la suppression de Pten stimulerait la mobilisation et la migration des HSC de la moelle osseuse vers la rate. Finallement, la transplantation de moelle osseuse délétée en Pten dans des souris immuno-déficientes montre que Pten fonctionnerait comme un suppresseur de tumeur dans le système hématopoiétique car son absence entraîne la formation rapide de leucémies lymphocytaires. Summary Identification, localization and activation of dormant hematopoietic stun cells in vivo Somatic stem cells are present in most self-renewing tissues including the skin, the intestinal epithelium and the hematopoietic system. On a single cell basis they have the capacity to produce more stem cells of the same phenotype (self-renewal) and to give rise to a defined set of mature differentiated progeny, responsible for the maintenance or repair of the host tissue. The best characterized adult stem cell is the hematopoietic stem cell (HSC) located in the bone marrow. One goal of my thesis work was to further characterize the location of endogenous HSCs in vivo. To do this, a technique called "label retaining assay» was used which takes advantage of the fact that stem cells (including HSCs) divide very infrequently and can be dormant for months. After labeling mice with the DNA analogue BrdU followed by a long BrdU free "chase", BrdU "label retaining cells" (CRCs) could be identified in the bone marrow. These CRCs were 300-fold enriched for phenotypic HSCs and by using flow cytometry analysis it could be shown that about 15% of all HSCs in the mouse are dormant for many weeks. Our results suggest that these long-term dormant HSCs are unlikely to be involved in homeostatic maintenance. However they are rapidly activated and reenter the cell cycle in response to injury signals such as myeloid ablation. In addition, detection of LRCs in combination with the HSC marker c-Kit could be used to locate engrafted dormant HSCs close to the endosteal lining of the bone marrow cavities. Most surprisingly, c-Kit+LRCs were found predominantly as single cells suggesting that the specific stem cell maintaining microenvironment, called niche, has limited space and may house only single HSCs. Complex roles of the tumor suppressor gene Pten in the hematopoietic system. The phosphatase PTEN is lost in hereditary and sporadic forms of human cancers, including gliomas, endometrial and breast cancers. Pten inhibits the PI3-kina.se pathway and plays a key role in apoptosis, cell growth, proliferation and migration. Our aim was to study the role of Pten in normal HSCs and during leukemia formation. To do this, we generated a mouse model in which the Pten gene can be deleted in hematopoietic cells including HSCs. This was achieved by crossing the conditional ptenflox allele with either the interferona inducible MxCre or the tamoxifen inducible Scl-CreERT transgene. This allowed the conversion of the ptenflox allele into a pterr' null allele in HSCs and other hematopoietic cell types. As a result Pten mutant mice developed massive splenomegaly due to a dramatic expansion of all myeloid cells. Interestingly, while the number of bone marrow HSCs progressively decreased, the number of HSCs in the spleen increased to a similar extent. Unexpectedly, extensive cell cycle analysis showed that Pten had little or no effect on HSC dormancy or HSC self-renewal. Instead, dramatically increased levels of the mobilizing cytokine G-CSF were detected in the blood serum suggesting that loss-of Pten stimulates mobilization and migration of HSC from the BM to the spleen. Finally, transplantation of Pten deficient BM cells into immuno-compromised mice showed that Pten can function as a tumor suppressor in the hematopoietic system and that its absence leads to the rapid formation of T cell leukemia.

Acetylated alpha-tubulin in Trypanosoma cruzi: immunocytochemical localization

We have used monoclonal antibodies specific for acetylated and non-acetylated alpha-tubulin to localize microtubules containing acetylated alpha-tubulin in all developmental forms of the life cycle of Trypanosoma cruzi. This was demonstrated using immunofluorescence and by transmission electron microscopy of thin sections, negative stained cells, and replicas of whole Triton X-100 extracted cells immunolabeled with antibody-gold complex. The antibody specific for acetylated alpha-tubulin (6-11B-1) binds to the flagellar, as well as to the sub-pellicular microtubules. The extent of labeling of the sub-pellicular microtubules with the monoclonal antibody recognized alpha-acetylated tubulin was smaller than that observed with the antibody which recognizes all tubulin isoforms. In relation to the developmental forms, the extent of labeling of the microtubules with antibody 6-11B-1 was larger in epimastigote and trypomastigote than in amastigote forms. Incubation of the parasites for 1 h at 0º C or in the presence of either colchicine or vinblastine did not interfere with the sub-pellicular microtubules. These observations, in agreement with those reported for Trypanosoma brucei brucei (Schneider et al., 1987; Schulze et al., 1987; Sasse per cent Gull, 1988) indicate that the sub-pellicular microtubules of trypanosomatids represent stable microtubules containing acetylated alpha-tubulin (or the alpha 3-tubulin isotype).

The spatio-temporal brain dynamics of processing and integrating sound localization cues in humans.

Interaural intensity and time differences (IID and ITD) are two binaural auditory cues for localizing sounds in space. This study investigated the spatio-temporal brain mechanisms for processing and integrating IID and ITD cues in humans. Auditory-evoked potentials were recorded, while subjects passively listened to noise bursts lateralized with IID, ITD or both cues simultaneously, as well as a more frequent centrally presented noise. In a separate psychophysical experiment, subjects actively discriminated lateralized from centrally presented stimuli. IID and ITD cues elicited different electric field topographies starting at approximately 75 ms post-stimulus onset, indicative of the engagement of distinct cortical networks. By contrast, no performance differences were observed between IID and ITD cues during the psychophysical experiment. Subjects did, however, respond significantly faster and more accurately when both cues were presented simultaneously. This performance facilitation exceeded predictions from probability summation, suggestive of interactions in neural processing of IID and ITD cues. Supra-additive neural response interactions as well as topographic modulations were indeed observed approximately 200 ms post-stimulus for the comparison of responses to the simultaneous presentation of both cues with the mean of those to separate IID and ITD cues. Source estimations revealed differential processing of IID and ITD cues initially within superior temporal cortices and also at later stages within temporo-parietal and inferior frontal cortices. Differences were principally in terms of hemispheric lateralization. The collective psychophysical and electrophysiological results support the hypothesis that IID and ITD cues are processed by distinct, but interacting, cortical networks that can in turn facilitate auditory localization.

Constitutive expression of a chimeric receptor that delivers IL-2/IL-15 signals allows antigen-independent proliferation of CD8+CD44high but not other T cells.

We have prepared transgenic mice whose T cells constitutively express a chimeric receptor combining extracellular human IL-4R and intracellular IL-2Rbeta segments. This receptor can transmit IL-2/IL-15-like signals in response to human, but not mouse, IL-4. We used these animals to explore to what extent functional IL-2R/IL-15R expression controls the capacity of T cells to proliferate in response to IL-2/IL-15-like signals. After activation with Con A, naive transgenic CD8+ and CD4+ T cells respond to human IL-4 as well as to IL-2. Without prior activation, they failed to proliferate in response to human IL-4, although human IL-4 did prolong their survival. Thus, IL-2-induced proliferation of activated T cells requires at least one other Ag-induced change apart from the induction of a functional IL-2R. However, a fraction of CD8+CD44high T cells proliferate in human IL-4 without antigenic stimulation or syngeneic feeder cells. In contrast, CD4+CD44high T cells are not constitutively responsive to human IL-4. We conclude that although all transgenic T cells express a functional chimeric receptor, only some CD8+CD44high T cells contain all molecules required for entry into the cell cycle in response to human IL-4 or IL-15.

Distinct subcellular localization of beta-tubulin epitopes in the adult mouse brain.

A panel of monoclonal antibodies specific of alpha-tubulin (TU-01, TU-09) and beta-tubulin (TU-06, TU-13) subunits was used to study the location of N-terminal structural domains of tubulin in adult mouse brain. The specificity of antibodies was confirmed b immunoblotting experiments. Immunohistochemical staining of vibratome sections from cerebral cortex, cerebellum, hippocampus, and corpus callosum showed that antibodies TU-01, TU-09, and TU-13 reacted with neuronal and glial cells and their processes, whereas the TU-06 antibody stained only the perikarya. Dendrites and axons were either unstained or their staining was very weak. As the TU-06 epitope is located on the N-terminal structural domain of beta-tubulin, the observed staining pattern cannot be interpreted as evidence of a distinct subcellular localization of beta-tubulin isotypes or known post-translational modifications. The limited distribution of the epitope could, rather, reflect differences between the conformations of tubulin molecules in microtubules of somata and neurites or, alternatively, a specific masking of the corresponding region on the N-terminal domain of beta-tubulin by interacting protein(s) in dendrites and axons.