Brain-derived neurotrophic factor (BDNF), which is sorted into a regulated secretory pathway of neurons, is supposed to act retrogradely through dendrites on presynaptic neurons or anterogradely through axons on postsynaptic neurons. Depending on which is the case, the pattern and direction of trafficking of BDNF in dendrites and axons are expected to be different. To address this issue, we analyzed movements of green fluorescent protein (GFP)-tagged BDNF in axons and dendrites of living cortical neurons by time-lapse imaging. In part of the experiments, the expression of BDNF tagged with cyan fluorescent protein (CFP) was compared with that of nerve growth factor (NGF) tagged with yellow fluorescent protein (YFP), to see whether fluorescent protein-tagged BDNF is expressed in a manner specific to this neurotrophin.We found that BDNF tagged with GFP or CFP was expressed in a punctated manner in dendrites and axons in about two-thirds of neurons into which plasmid cDNAs had been injected, while NGF tagged with GFP or YFP was diffusely expressed even in dendrites in about 70% of the plasmid-injected neurons. In neurons in which BDNF-GFP was expressed as vesicular puncta in axons, 59 and 23% of the puncta were moving rapidly in the anterograde and retrograde directions, respectively. On the other hand, 64% of BDNF-GFP puncta in dendrites did not move at all or fluttered back and forth within a short distance. The rest of the puncta in dendrites were moving relatively smoothly in either direction, but their mean velocity of transport, 0.47 ± 0.23 (SD) μm/s, was slower than that of the moving puncta in axons (0.73 ± 0.26 μm/s).The present results show that the pattern and velocity of the trafficking of fluorescence protein-tagged BDNF are different between axons and dendrites, and suggest that the anterograde transport in axons may be the dominant stream of BDNF to release sites. Neurotrophins have been considered to play roles in the differentiation, neurite outgrowth and survival of a certain group of neurons -4. In adtrans-Golgi network, and transported to release sites in neurites [in situ hybridization technique after the fixation of neurons, or by observing the decrease in fluorescence intensity of neurons expressing BDNF tagged with green fluorescent protein (GFP), the actual dynamics of BDNF trafficking was not analyzed in dendrites. Thus, a question of whether the trafficking of BDNF is different between axons and dendrites of neurons is not answered yet.To serve such a broad-ranging function, BDNF produced in the nucleus of neurons is sorted into a regulated secretory pathway through the neurites ,11. In aneurites -15. In dneurites -22. SincAn answer to this question will give a clue that may resolve the controversial issue of whether BDNF acts retrogradely through dendrites on presynaptic neurons or anterogradely through axons on postsynaptic neurons. To address this question, it is desirable to perform a real-time analysis of movements of BDNF tagged with GFP in both axons and dendrites of living neurons. However, such an analysis of BDNF trafficking has not been successfully carried out except for two recent studies on its retrograde transport in dorsal root ganglion neurons  and bidiIn the present study we carried out a real-time analysis of movements of BDNF tagged with GFP in both axons and dendrites of living cortical neurons using the method of direct injection of their plasmid cDNAs into the nucleus . We founParts of the present results were published in an abstract form .Plasmids encoding BDNF tagged with GFP or CFP at the COOH-terminus were injected into the nucleus of cultured cortical neurons through a micropipette under visual control, as reported previously . GFP- orIn about two-thirds of the neurons that expressed fluorescent signals of BDNF-GFP, the signals were punctated in clusters along dendrites and axons Fig. . The somIn the present study we attempted to inject plasmids into pyramidal cell-like neurons which are excitatory. To confirm that neurons expressing fluorescent signals were excitatory, they were stained immunocytochemically with anti-glutamic acid decarboxylase 65 (GAD65) antibody. As shown in Figure In part of the experiments we injected plasmids encoding NGF-GFP to see whether BDNF-GFP is expressed in a manner specific to this neurotrophin. The granular expression of NGF-GFP was seen in a minor group of the cells that expressed fluorescent signal see Fig. . In mostAs mentioned before, the single injection of plasmid cDNAs encoding GFP-, CFP- or YFP-tagged neurotrophins also showed the difference in the expression pattern between BDNF and NGF Fig. . In thisWe then found that most of the fluorescent puncta were moving in neurites of living neurons in the anterograde as well as retrograde directions .125I-labelled BDNF in the optic nerve of neonatal rats  is comparable to the reported values for the anterograde transport of a synaptic vesicle protein, synaptophysin tagged with GFP, in the axons of mouse dorsal root ganglion cells . The pr92 μm/s,  and that92 μm/s, . The que125I-labelled BDNF in the optic nerve of neonatal rats. This suggests that the trafficking properties of BDNF-GFP are not significantly different from those of endogenous BDNF. Furthermore we observed that the expression pattern of BDNF-GFP or -CFP was different from that of NGF-GFP or -YFP in most cases. These results altogether suggest that the expression and trafficking of BDNF-GFP may not be substantially different from those of endogenous BDNF. This further suggests that the GFP tag may not notably affect sorting and trafficking of secretory proteins, as reported previously [In the present study using the plasmid cDNA injection method, there is a possibility that BDNF-GFP was expressed at much higher level than that of endogenous BDNF, and thus the sorting and trafficking characteristics would be different from those of endogenous BDNF. Although we cannot completely exclude this possibility, we believe that it is unlikely, based on the two observations. 1) The intracellular localization of expressed BDNF-GFP is similar to that of endogenous BDNF when BDNF-GFP is expressed in a punctated manner. 2) As discussed above, the mean velocity of trafficking of BDNF-GFP in axons is similar to the reported value for the anterograde transport of eviously .In sum, the present finding that the anterograde trafficking of BDNF-GFP in axons was dominant in cortical neurons seems consistent with the view that BDNF acts on postsynaptic neurons in the anterograde direction -15,29-35The present results show that the pattern and velocity of the trafficking of BDNF are different between the two structural domains of neurons, axons and dendrites, and suggest that the anterograde transport in axons may be the dominant stream of BDNF to release sites.Neonatal mice  were anesthetized with ketamine , and then killed by cervical dislocation. The experimental procedures met the regulation of the Animal Care Committee of Osaka University Graduate School of Medicine. Neurons were cultured at a low density using conventional methods, as reported previously . A pieceThe cDNAs of mouse BDNF or NGF tagged with GFP, CFP or YFP at the C-terminus were provided by Dr. Masami Kojima. Glass micropipettes were filled with Tris-ethylene-diamine-tetraacetic acid (EDTA) buffer (pH 8.0) which contained cDNAs of BDNF-GFP, or -CFP (0.5–1 μg/μl), or NGF-GFP, or -YFP (0.5 μg/μl). Then cultured neurons were placed on the stage of an inverted epifluorescence microscope , and cDNAs were injected into the nucleus of a neuron through a micropipette under visual control, using a micromanipulator , as described previously .Neurons which expressed fluorescent protein-tagged BDNF or NGF signals were observed using a 40 X /1.3 NA oil immersion objective  attached to the inverted epifluorescence microscope. The fluorescence of GFP. CFP and YFP excited by light at the wavelength of 488, 434 and 514 nm was measured using a cooled CCD camera . Time-lapse recordings were carried out about 24 h after the transfection, and sequential images were acquired using a cooled CCD camera at about 30°C. The exposure time was 1 or 2 s, the lapse of time was about 5 s, and the total time of recordings was 15–20 min. The trafficking of BDNF was analyzed by tracking the positions of GFP fluorescence-positive vesicles in neurites as a function of time. The analyses and animations of the data were made using the AQUA COSMOS software (Hamamatsu Photonics).For immunocytochemical staining, neurons were fixed with 4% paraformaldehyde  and 4% sucrose in Dulbecco's phosphate-buffered saline (PBS) for 20 min at room temperature. The cells were incubated with PBS containing 0.2% Triton-X (Sigma) for 5 min and blocked by 10% goat serum in PBS for 1 h at 37°. Then, anti-MAP2 monoclonal antibody , anti-BDNF polyclonal antibody , and anti-GAD65 monoclonal antibody  were applied overnight at 4°. Endogenous BDNF was visualized by anti-rabbit secondary antibody conjugated with Alexa 546 . MAP2 and GAD65 were visualized by anti-mouse secondary antibody conjugated with Alexa 647 or 350 , and by isotype-specific secondary antibody conjugated with Alexa 546 .BDNF, brain-derived neurotrophic factorCFP, cyan fluorescent proteinGAD65, glutamic acid decaroxylase 65GFP, green fluorescent proteinKIF, kinesin superfamily proteinMAP2, microtubule-associated protein 2NGF, nerve growth factorPBS, phosphate-buffered salineYFP, yellow fluorescent proteinNA designed and carried out most of the experiments. KH helped with the design and analysis of experiments. TT planned the experiments and coordinated the work.Supplementary movie of BDNF-GFP puncta in an axon and dendrites of a cortical neuron. An arrow at rest indicates an example of non-moving and fluttering puncta in dendrites, and another moving arrow that appeared 6 min after starting the movie indicates an example of fast-moving puncta in axons.Click here for file


The purpose of this study was to examine whether perinatal exposure to two major environmental endocrine-disrupting chemicals, bisphenol A  and nonylphenol  daily from gestational day 3 to postnatal day 20  would lead to behavioral alterations in the male offspring of F344 rats. Neither BPA nor NP exposure affected behavioral characteristics in an open-field test (8 weeks of age), in a measurement of spontaneous motor activity (12 weeks of age), or in an elevated plus-maze test (14 weeks of age). A passive avoidance test (13 weeks of age) showed that both BPA- and NP-treated offspring tended to delay entry into a dark compartment. An active avoidance test at 15 weeks of age revealed that BPA-treated offspring showed significantly fewer avoidance responses and low-dose NP-treated offspring exhibited slightly fewer avoidance responses. Furthermore, BPA-treated offspring significantly increased the number of failures to avoid electrical unconditioned stimuli within 5-sec electrical shock presentation compared with the control offspring. In a monoamine-disruption test using 5 mg/kg  tranylcypromine (Tcy), a monoamine oxidase inhibitor, both BPA-treated and low-dose NP-treated offspring at 22–24 weeks of age failed to show a significant increment in locomotion in response to Tcy, whereas control and high-dose NP-treated offspring significantly increased locomotion behavior after Tcy injection. In addition, when only saline was injected during a monoamine-disruption test, low-dose NP-treated offspring showed frequent rearing compared with the control offspring. The present results indicate that perinatal low-dose BPA or NP exposure irreversibly influenced the reception of fear-provoking stimuli , as well as monoaminergic neural pathways. Recently, there has been increasing concern about the exposure of the developing fetus to environmental endocrine-disrupting chemicals (EDCs). The disruption of cognitive function and various behavioral traits due to EDC exposure has been suspected  because in vitro and in vivo assays. More recent studies have also identified BPA as an antiandrogen by a yeast two-hybrid system  is a high-production4volume chemical used in the manufacture of polycarbonate plastics, epoxy resins, and polyester resins. Worldwide production of BPA has increased and will most likely continue to increase in the future . Human ed system  and as ad system . These vin vitro and in vivo assays (in vivo conditions.NP (4-nonylphenol) is another environmental EDC with week estrogenic activity . NP is uo assays . Althougo assays . NP may There have been a number of reports suggesting the adverse effects of perinatal exposure to BPA on various behavioral traits in laboratory rodents. In mice, exposure to BPA during fetal development was shown to alter maternal behavior  and enhaIn contrast, only a few studies have reported the effects of perinatal NP exposure on the behavioral traits of the offspring of experimental animals. In the present study, we examined the adverse effects of low-dose (0.1 mg/kg/day) perinatal BPA or NP exposure on behavioral characteristics. To this end, we performed a series of behavioral tests: an open-field test, a measurement of spontaneous activity during a dark phase, a step-through passive avoidance test, an elevated plus-maze test, and a two-way shuttle-box avoidance test. In addition, in order to evaluate suspected alterations in the monoaminergic system, we investigated behavioral responses to tranylcypromine (Tcy), a monoamine oxidase inhibitor.n = 10–11/condition). The dams were orally exposed to BPA  or NP  dissolved in corn oil, or to corn oil alone  from GD3 until postnatal day (PND)20. Oral administrations of BPA and NP were performed by gavage. Because animals were trained to receive the feeding needle before mating, this procedure was not stressful. The dams were examined for clinical signs of toxicity and were weighed daily before dosing. After parturition (PND0), the pups were counted, weighed, and assigned to groups of six pups per litter, maintaining equivalent sex distributions when possible. Pups remained with their biological mother. Offspring were weighed and the body weights recorded on PND0, 3, 7, 10, 14, and 21 and again at 8 and 13 weeks of age. We included the mean weight of each littermate in the statistical analysis. Male pups were marked with ink for identification; On PND21, the marked male pups were gathered from different litters and housed together according to treatment group (7–8/cage). The dams were anesthetized with diethyl ether and then sacrificed by exsanguination; the body weights and organ weights  were then recorded.Male and female F344/N rats were purchased from SLC . The animals were maintained under controlled temperature (24 ± 1°C) and humidity (55 ± 5%), on a 12-hr light (09:00–21:00 hr):12-hr dark (21:00–09:00 hr) cycle. Food and water were freely available. After acclimatization for 1 week, female rats were placed with males. Vaginal smears were examined daily: a sperm-positive smear determined gestational day (GD)0. After detection, the pregnant dams were housed individually and were randomly assigned to an exposure condition (n = 9–10/group). The remaining male pups in the litter were subjected to the measurement of organ weights  at weaning (PND21) or at 8 weeks of age. Although male rats were usually housed in groups according to experimental treatment, rats were housed individually for some behavioral tests. At the end of each behavioral test, rats were again housed in a group according to treatment. In this study, we excluded female pups from behavioral tests because the estrous cycle in mature females affects various behavioral characteristics. When using female animals, it is important to consider the estrous cycle in evaluating the results of behavioral tests that require several consecutive days. This study was approved by the Animal Care and Use Committee of the Graduate School of Agricultural and Life Sciences, University of Tokyo.We randomly selected one male pup per litter to undergo a series of behavioral tests ; none of the animals was familiar with this apparatus. Open-field behavior was recorded for 5 min by a video camera positioned above the apparatus during the dark phase (21:30–23:00 hr) under a low white light; responses were automatically analyzed by a computer-assisted system, which classified observed behavior as locomotion, rearing, or “other” behaviors.We measured spontaneous motor activity in 12-week-old male offspring using a Supermex  . The SupWe conducted the step-through passive avoidance test when the animals were 13 weeks of age. The test was carried out during the light phase (13:00–17:00 hr), and each animal was housed individually during the test. The passive avoidance apparatus consisted of light and dark compartments. The first time each animal was placed in this apparatus, an electric foot shock  was delivered to the animal through the grid floor just after the animal had completely left the light compartment for the dark compartment. We recorded the latency period required before each animal entered the dark compartment after having been placed in the light compartment. Twenty-four hours later, a retention trial (with no shock) was performed, and the latency period before entering the dark compartment was recorded. In addition to the traditional measure, we recorded the frequency and percentage of duration of poking into the dark compartment until the animal completely entered the dark compartment in the retention trial. If an animal failed to enter the dark compartment within 20 min, the test was terminated.The elevated plus-maze apparatus consisted of two open arms (50 × 10 cm) and two closed arms  extending from a central square platform (10 × 10 cm); arms were arranged so that those of the same type were opposite each other. The apparatus was elevated 60 cm above the floor. At 14 weeks of age, each animal was placed in the central square facing an open arm during the dark phase (21:30–23:00 hr). We then recorded standard spatiotemporal factors for 5 min .At 15 weeks of age, animals were subjected to an active avoidance test in a two-way shuttle-box  consisting of two compartments connected to each other by a hole in the wall; this test was carried out during the light phase (13:00–17:00 hr). Each animal was housed individually through the active avoidance test. Each animal was allowed to become accustomed to the shuttle-box apparatus for 5 min before every session; the animal was then subjected to 25 daily trials/session of avoidance conditioning in four consecutive sessions (acquisition test). For each trial, a 5-sec conditioned stimulus (CS), consisting of a buzzer and light, was followed by a 5-sec unconditioned stimulus (UCS), which included a scrambled shock of 0.2 mA delivered through the floor grid. In addition, on the day after the fourth session, each rat performed the extinction test, which is basically the avoidance text without the UCS. Each trial was separated by variable intertrial intervals . During the acquisition tests in sessions 1–4 and the extinction test, we recorded the percentage of correct avoidance responses, in which the animals moved to the other compartment of the shuttle box within a 5-sec CS in each block of 25 trials. To evaluate further behavioral characteristics in this procedure, we recorded the percentage of failures to avoid the stimulus within 5-sec UCS and the latency periods associated with both the CS and UCS throughout the four acquisition sessions.trans-2-phenylcyclopropyl-amine hydrochloride; Sigma-Aldrich, St. Louis, MO, USA). At 22–24 weeks of age, BPA- or NP-treated male offspring were subjected to the monoamine-disruption test. Before the monoamine-disruption tests, we determined the optimal dose of Tcy for the monoamine-disruption test in a different set of male F344 rats (n = 15) at 9 weeks of age. We injected (i.p.) Tcy solution in 0.9% saline at 0, 2, 5, and 10 mg/mL (1 mL/kg) at 16:00 hr  and then measured spontaneous motor activity as described above. We confirmed that animals treated with 5 mg/kg Tcy showed a high increment of activity at 21:30 hr, when open-field behavior was recorded.Disturbances of the monoaminergic system in the CNS were induced by a single intraperitoneal (i.p.) injection of Tcy  as a vehicle into each rat; 5.5 hr after the injection, we observed and recorded the behavior of the animals in the open-field apparatus for 4 min.On the day after the saline challenge, we injected 5 mg/kg Tcy (i.p.) into the same animal and recorded open-field behavior for 4 min, as described for the saline challenge. Behavioral analyses in the monoamine-disruption test were performed as described for the open-field test.p < 0.05.We conducted statistical analyses using StatView, Version 5.0 . We analyzed the effects of perinatal BPA or NP exposure on maternal body weight increase and the body weight of male offspring by analysis of variance (ANOVA) with one between-subject factor (treatment) and one repeated-measures factor (days). The number of total, male, and female pups;, the organ weights of dams at weaning; and the organ weights of male offspring at PND21 and at 8 weeks of age were analyzed by one-way ANOVA. Behavioral measurements were analyzed by one-way ANOVA, except for the percentages of correct avoidance in the shuttle-box avoidance test, which were assessed by repeated measures of ANOVA over days (sessions). In the analysis of latency in the passive avoidance test, data processed through logarithmic transformations were used for the ANOVA because of their significantly inappropriate distributions with respect to the normal distributions. In each statistical analysis, the effects of BPA and NP exposure were analyzed with respect to the control in the same ANOVA. When the ANOVA produced significant results, we then performed the post hoc Fisher’s protected least-significant difference test for comparisons between groups. The significance level for all tests was set at Oral exposure to BPA or NP showed no statistically significant effect on maternal body weight increase during pregnancy and lactation or on the number of total, male, and female pups (data not shown). All dams in this study delivered their offspring on GD22. There was no significant effect of 40-day exposure to BPA or NP on either body weight or organ weights (data not shown).Perinatal exposure to BPA or NP had no significant effect on either body weight gain or organ weights of male offspring on PND21 or at 8 weeks of age (data not shown). No male offspring died during the course of the study (> 25 weeks of age).F = 0.271, p > 0.5] or the number of rearings .In the open-field test, neither BPA nor NP exposure significantly affected the percentage of locomotion , or the immobile time  during the 12-hr dark phase (data not shown).To assess general motor activity under nonstress conditions, we recorded spontaneous motor activity of male offspring. Neither BPA nor NP exposure had any effect on the rhythm of activity, the total counts of activity  and multiple comparisons revealed that the subjects showed significant hesitation (p < 0.01) to enter the dark compartment compared with the short latency during shock presentation in all of the experimental groups  = 1.166, p > 0.1; F = 1.919, p > 0.1; On shock-presenting day of the passive avoidance test, the subjects readily entered the dark compartment (< 30 sec). During the retention trial 24 hr after shock presentation, ANOVA  or the closed arms  of the elevated plus-maze test, although the frequency of entering the open arms was slightly higher in the BPA-treated group than in the controls (data not shown).Neither BPA nor NP exposure significantly altered the frequency of entering the open arms  and number of sessions , as well as a significant interaction between chemical exposure and the number of sessions . One-way ANOVAs and post hoc multiple comparisons for each session indicated significantly fewer avoidance responses in BPA-treated offspring at the first, second, and third sessions than in the control offspring  = 0.571, p = 0.104]. One-way ANOVA indicated a significant effect of chemical exposure and that the frequency of failure of avoidance within 5 sec of shock presentation—in which one-way ANOVA indicated a significant effect of chemical exposure —in BPA-treated offspring was significantly higher (p < 0.001) than that in the control offspring; low-dose NP-treated offspring showed a similar tendency  = 0.722, p > 0.5; F = 1.186, p > 0.1; BPA and low-dose NP exposure significantly affected the avoidance responses of the male offspring in the active avoidance test. Repeated-measures one-way ANOVA showed a significant effect of chemical exposure  and the number of rearing behaviors  yielded the following results. In the monoamine-disruption test, control and high-dose NP-treated offspring showed a significant increase in locomotion behavior resulting from Tcy injection . Tcy also caused significant decreases in the number of rearing behaviors in all experimental groups (p < 0.1), which was abolished by monoamine disruption by Tcy , and general development (body weight and organ weights) of the offspring confirmed that there were no adverse effects of BPA at 0.1 mg/kg/day or of NP at 0.1 and 10 mg/kg/day, which was consistent with the findings of previous reports .In the present study, we detected no statistically significant alterations by BPA or NP in the spontaneous activity and behaviors of rats in the open-field test at 8 weeks of age and in the elevated plus-maze test at 14 weeks of age. This suggests that these chemical exposures induce no severe abnormalities in general behavior.In the passive avoidance test, offspring perinatally exposed to BPA or NP seemed to be more sensitive to fear-inducing shock than were the control offspring, which might have led to the somewhat stronger retention in the chemical-treated groups; however, such changes were statistically ambiguous because of the large individual differences in the experimental conditions used in this study.In the active avoidance test, BPA and low-dose NP showed clear or partial adverse effects on behavior, respectively. In particular, BPA-treated offspring may have been less able to learn than the control offspring in terms of causality. Low-dose NP-treated offspring were also affected to some extent. Although the possibility remains that BPA-treated offspring were more insensitive to electrical shock than were the control offspring, the slight elongation of the latency period in the passive avoidance test would have excluded this possibility. If BPA-treated offspring had found the electrical stimuli less fear-provoking and/or painful, they would have entered the dark compartment more quickly than the control offspring in the passive avoidance test. It would also be unlikely that less learning took place as a result of motor dysfunction or sensory abnormality because there was no alteration in the spontaneous activity during the dark phase and in the duration of locomotion in the open-field test, as well as in the latency periods associated with CS and UCS in the active avoidance test. When electrical shock was presented as a UCS, BPA-treated offspring failed to enter the opposite compartment within 5 sec more frequently than the control offspring, and NP-treated offspring showed a similar tendency. BPA-treated offspring tended to stiffen in the corner of the box during the UCS, and these animals appeared to stop avoiding the UCS more often than did the control offspring, as determined by direct observation (data not shown). It is possible that excessive fear of the UCS would interfere with the smooth progression of avoidance learning. Perinatal BPA exposure may render male offspring exceedingly vulnerable to intolerable levels of fear. Interestingly, this hypothesis may be supported by a previous study , which iIn the monoamine-disruption test, Tcy-induced increases in locomotion were significantly less marked in BPA-treated and low-dose NP-treated offspring, but not in the high-dose NP-treated offspring, compared with the control offspring. This is the first study reporting behavioral alterations due to perinatal BPA and NP exposure shown by responses to the disruption of monoaminergic systems, with Tcy having clear and straightforward pharmacologic effects as a monoamine oxidase inhibitor. Previous studies have reported changes after BPA exposure in a mouse model of psycho-stimulant abuse (methamphetamine)  and in aWe cannot address differences in sexes in the present study because we limited this behavioral study to the male offspring; our primary goal was to detect behavioral alteration by perinatal BPA exposure in male offspring. However, studies in rats by In the present study, one animal per litter sequentially underwent all of the behavioral tests. It is possible that an experience in an earlier behavioral test influenced the results of the subsequent behavioral tests. For example, a painful experience immediately after exploratory behavior in the passive avoidance test might interfere with the behavioral propensity in the elevated plus-maze test. However, we believed that using sequential behavioral tests in the same animal would not obstruct the evaluation of effects of perinatal exposure to chemicals because all of the animals in the four treatment groups experienced the stimuli.in vitro and in vivo estrogenic potency of these compounds determined by certain simple assay systems. It may be useful to consider other potencies and/or metabolites of BPA and NP (In summary, perinatal exposure to BPA and NP, both at 0.1 mg/kg/day, affected the extent of shock-related behavior and affected responses to the disruption of the mono-aminergic system, although the direct mechanisms of these alterations remain unclear at present. Moreover, the neurobehavioral toxicity of both BPA and NP may be out of proportion with the A and NP  in additA and NP . In our A and NP , BPA andWe conclude that perinatal BPA and NP exposure, even at slightly higher doses than those associated with environmental exposure in humans, had adverse behavioral effects on rats, especially when the animals were forced to avoid fear-provoking stimuli such as electrical shocks. Perinatal exposure to BPA and NP disrupted the reception of intolerable stress, which may be due to the alterations in monoaminergic system.


Studies of gene expression profiling have been successfully used for the identification of molecules to be employed as potential prognosticators. In analogy with gene expression profiling, we have recently proposed a novel method to identify the immunophenotypic signature of B-cell chronic lymphocytic leukemia subsets with different prognosis, named surface-antigen expression profiling. According to this approach, surface marker expression data can be analysed by data mining tools identical to those employed in gene expression profiling studies, including unsupervised and supervised algorithms, with the aim of identifying the immunophenotypic signature of B-cell chronic lymphocytic leukemia subsets with different prognosis. Here we provide an overview of the overall strategy employed for the development of such an "outcome class-predictor" based on surface-antigen expression signatures. In addition, we will also discuss how to transfer the obtained information into the routine clinical practice by providing a flow-chart indicating how to select the most relevant antigens and build-up a prognostic scoring system by weighing each antigen according to its predictive power. Although referred to B-cell chronic lymphocytic leukemia, the methodology discussed here can be also useful in the study of diseases other than B-cell chronic lymphocytic leukemia, when the purpose is to identify novel prognostic determinants. B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease with highly variable clinical courses. Two major clinical staging systems, mainly based on tumor load, were developed to estimate prognosis in B-CLL -3. Both H genes has recently been identified as a robust indicator of disease outcome: patients with a disease characterized by neoplastic cells bearing a mutated IgVH gene configuration had significantly longer survival than those cases affected by B-CLL expressing unmutated IgVH genes. Since IgVH mutation testing is an expensive and technically difficult assay not widely applicable for clinical use, subsequent studies were focused on the identification of alternative markers with prognostic value similar to that of IgVH mutations, and whose expression could easily be investigated, e.g. by flow cytometry. Several reports identified the over-expression of CD38 as a marker of poor prognosis for B-CLL patients [H mutational status, initially emphasized [The mutational status of IgVpatients . Howeverpatients -6., and patients . Moreovephasized -6,9.H mutations [Studies of gene expression profiling (GEP) have been successfully used for the identification of additional molecules to be employed as potential prognosticators -13. Amonutations ,14-16. TIn analogy with GEP, we have recently proposed a novel method to identify the immunophenotypic signature of B-CLL subsets with different prognosis, named surface-antigen expression profiling (SEP),18. In oWe will provide here an overview of the strategy employed for the development of this sort of "outcome class-predictor" for B-CLL based on surface-antigen expression. In particular, we will discuss how to compute flow cytometry data, the rationale for the choice of sequential unsupervised/supervised analyses eventually yielding to the signature of the identified disease subsets. Finally, we will also discuss how to transfer the information gained through the proposed class-predictor into the routine clinical procedures to refine the identification of B-CLL patients with different prognosis. In particular, we will summarize a flow-chart indicating how to select the immunophenotypic markers with the most relevant prognostic impact and how to build-up a prognostic scoring system by giving different weights to each antigen according to its predictive power. Part of the data extensively discussed in this section of the present review has been recently reported by our group .All the comments and analyses reported below, although referred to B-CLLs, can be useful to transfer a similar approach into the study of diseases other than B-CLL when the aim is of identifying novel prognostic determinants.+ B-CLL cells displaying a specific fluorescence intensity greater than the 98–99% of the same cell population stained with isotype- and fluorochrome-matched control immunoglobulins [Expression of surface antigens is usually investigated by multicolor flow cytometry . In our lobulins ,24,25. Ilobulins :advantages for the subsequent analyses: in our hands the use of percent of positive cells allowed a better application of the methods of cluster analyses reported below; this happens basically for two reasons: i) it facilitates clustering by decreasing the complexity of the final database ; ii) it reduces the operational range of the color scale employed in heat maps .- advantages for the recruitment of cases: an absolute measuring of flow cytometry data, e.g. by converting MFI values into molecules equivalent of soluble fluorochrome (MESF) [- e (MESF) , can be advantages due to the characteristics of the chosen panel: the use of percent of positive cells as a measure of antigen expression allows to better compare all the employed antigens to each other especially when the panel of interest: i) includes mAbs conjugated with fluorochromes with different relative brightness ; ii) includes a certain number of unlabeled mAbs, which, by requiring additional binding with secondary fluorochrome-conjugated antibodies, usually yield higher MFI values than directly fluorochrome-conjugated mAbs; iii) includes mAbs recognizing phenotypic markers known to have greatly different cellular density as compared to others  [- nd CD59) ,18,27.Last but not least, this choice of analysis might be prospectively more useful in the field of B-CLL for two additional reasons: i) compare the expression of some markers of supposed prognostic significance with that of other well-established prognosticators, e.g. CD38 or ZAP-70, whose expression level is usually reported as percent of positive cells -16,24,28If the working hypothesis is to analyse surface antigen expression data in order to identify disease subsets characterized by a given expression pattern, we need computational tools capable to simultaneously compare expression levels of the various antigens among different cell samples.In our B-CLL studies ,18., floThe chosen statistical and computational tools of pattern recognition/classification are those normally employed in GEP studies ,20. So fIn our original proposal on B-CLL , we haveOperationally, in our original report , we analThe sequential use of hierarchical clustering and k-means to eventually define the SEP of a given number of disease subsets (in our example three B-CLL subsets) has its rationale in the specific mathematical algorithms underlying these two unsupervised methods. In particular, hierarchical clustering is usually utilized in preliminary screening, when no further information is available; in our example, hierarchical clustering of B-CLLs, if associated with Kaplan-Meier analysis , was ablThe three B-CLL clusters identified by the sequential application of hierarchical and k-means analyses  were prIn our original studies, the combination of unsupervised analyses  and Kaplan-Meier survival curves, allowed the identification of three phenotypic clusters in B-CLLs, one corresponding to a good prognosis B-CLL subset, the remaining two clusters characterized by shorter median survivals, although with different immunophenotypic profiles .The next logical step is the identification of the phenotypic signature, i.e. the minimal number of surface markers succinctly characterizing each B-CLL prognostic group. For this purpose, we chose to apply the "nearest shrunken centroid" algorithm, as proposed by Tibshirani et al ,20., utiThe "nearest shrunken centroid" classification makes one important modification to standard "nearest centroid" classification. For a given antigen, it shrinks each centroid corresponding to each subgroup toward the overall centroid  by a fixed amount, called "threshold". This shrinkage consists of moving the centroid towards zero by a value corresponding to the threshold, setting it equal to zero if it hits zero. After shrinking the centroids, the new sample is classified by the usual nearest centroid rule, but using the shrunken centroids. This shrinkage has the operational advantages (i) to make the classifier more accurate by reducing the effect of noisy antigens, and (ii) to do an automatic selection of the antigens. In particular, if a given antigen is shrunk to zero for all the subgroups, then it is eliminated from the prediction rule. Alternatively, it may be set to zero for all subgroups except one; in this latter case, we learn that above- or below-average expression for that antigen characterizes that subgroup.In the "nearest shrunken centroid" method, the user decides on the value for threshold to be employed. Typically, different choices are examined, and to guide the user to this choice, PAM performs k-fold  cross-validation (CV) for a range of threshold values. Basically, a model is fitted on 90% of the sample and the class of the remaining 10% is predicted; this procedure is repeated 10 times, with each sample being part of the 10% utilized as tester at least once. The overall error is obtained by summing the errors of all the 10 parts added together. This procedure is done for a series of threshold values, and the number of CV misclassification errors for each threshold level allows the user to choose the value giving the minimum cross-validated misclassification error rate.In the case of our original B-CLL studies, we randomly divided immunophenotypic data (123 cases) into training data (90 cases) and test data (33 cases) ; data frA list of the 12 selected markers overall representing the immunophenotypic signature of the three B-CLL subsets with different prognosis are reported in Fig. H mutations and ZAP-70 expression as well as the biological implication underlying differential expression of critical molecules in the newly identified B-CLL subsets have been extensively discussed elsewhere [Even though information regarding correlations with IgVlsewhere , we brieH mutations and ZAP-70 expression have been reported to be among the most important prognosticators for B-CLL [H mutations below the established cut-off of 2% and expression of ZAP-70 in more than 20% of neoplastic cells had a negative impact on survivals also in our series [H gene configuration or expressed ZAP-70 in less than 20% of cells frequently belonged to the immunophenotypic group characterized by the best prognosis (group I); conversely, patients characterized by B-CLL cells lacking IgVH mutations or with high ZAP-70 expression levels belonged more frequently to the immunophenotypic groups associated with worse prognosis (groups II and III) [IgVor B-CLL ,28,40-42r series . Consistand III) .+CD54+CD25+ phenotype more frequently displayed high number of IgVH mutations [H mutational status consistent with antigen-driven selection [H mutations usually occur as the result of T cell-dependent interactions during GC maturation of B cells [According to the presented results, overexpression of CD62L, CD54, CD49c and CD25 in the absence of CD38 represented the immunophenotypic signature of good prognosis B-CLL. Noteworthy CD62L, together with CD54 and CD25 are all surface structures somewhat involved in the cross-talk between B lymphocytes with neighbouring endothelial and/or T cells within the lymph node microenvironment -47. Inteutations ,40-42, aelection ,49. Thes B cells .+ from CD38- B-CLL cells [H mutation load [One of the two immunophenotypic profiles characterized by worse prognosis (group II) was distinguished by the above-average expression of CD38, CD49d, CD29 and CD49e. This finding is in keeping with recent reports in which high levels of CD49d mRNA and protein were found to be part of the signature distinguishing CD38LL cells ,52. The LL cells , as wellLL cells -56. Alonion load -16,28,40H percent mutations and/or lack of antigen-driven selection [H mutations capable to split B-CLL patients into subgroups with different survivals [A third immunophenotypic profile (group III), associated with poor prognosis and succinctly characterized by the below-average expression of all the markers reported above as over-expressed in group I  or group II  B-CLLs, was revealed by supervised analysis . The ideelection ,48,49; telection )), that urvivals .As summarized in Fig. In the present paragraph, we discussed the overall strategy allowing the selection, among the markers identifying each subset, of those with the most relevant prognostic impact to be eventually used for prognostic purposes in clinically-oriented laboratories. Such a strategy included at least two initial steps aimed (i) at reducing the number of prognosticators by keeping only the most relevant of them, and (ii) at finding the optimal cut-off points to be employed to discriminate cases expressing or not a given marker. In the case of B-CLLs, we operated as follows:This first point was addressed by applying the Cox proportional hazards regression model on expression data for the 12 antigens with overall survival as dependent variable . The notThe Cox proportional hazards regression model computes a coefficient (z score) for each predictor marker that indicates the direction and degree of flexing that the predictor has on the survival curve. Zero means that a given marker has no effect on the curve, i.e. it is not a predictor at all; a positive z score indicates that larger values of the marker are associated with greater mortality, while a negative z score indicates that larger values of the variable are associated with lesser mortality.A list of the actual z scores, as found by us in a series of 137 B-CLLs  are repoOnce identified the phenotypic markers with greater prognostic impact, we operated to find, for each of them, the optimal cut-off value yielding the best separation between two subgroups with different survival probabilities.In general it can be assumed that any given prognosticator  allows for a classification of patients into groups with different risk with respect to a response variable . The functional relationship between the putative prognosticator and the response variable is unknown. Any given cut-off point for the expression level of an antigen determines two groups of patients, i.e. a group with all the patients in which the variable is less or equal to a given cut-off point, and a group of patients in which the variable is greater than the same cut-off point. The determination of the optimal cut-off point for each antigen yielding the best separation between two subgroups with different survival probabilities was achieved by applying the maximally selected log-rank statistics , availabH mutations in studies aimed at testing the strength of these markers as prognosticators [As reported in Figs. ticators ,63,64.Taken together, univariate Cox proportional hazard regression and maximally selected log-rank statistics provide useful information on the prognostic value of each single antigen, when considered alone. As a next step, we tried to improve the prognostic assessment of B-CLL by combining the expression values of the six antigens into a comprehensive prognostic score. To do this, we operated as follows Figs.  and 3:Scores of "0", "1" and "2" were assigned to each prognosticator according to the z score found for each antigen and the expression above or below the established cut-off value. In particular, for positive prognosticators  a score "0" was assigned when the expression was below the cut-off values, while for negative prognosticators  a score "0" was assigned when the expression was above the established cut-offs. Scores "1" were assigned to the positive prognosticators CD49c and CD54 when the expression was above the established cut-offs, and to the negative prognosticator CD79b when the expression was below the established cut-off. These markers were characterized by z scores comprised between the absolute values of 2 and 3 . We therefore labeled the identified three prognostic groups as high-, intermediate- and low- risk groups, respectively.The final step is the sum of the values found for each prognosticator by considering the assigned scores , as defined above. In a series of 115 B-CLL patients we obtained total score values ranging from "0" (complete absence of phenotypic conditions associated with good prognosis) up to "9" . Overall, the 115 B-CLL patients showed a median survival time of 157 months with 95% confidence intervals ranging from 120 months to "not reached" . The samFig. By summarizing some recent studies by our group ,18,22, wThe approach proposed and discussed in the present review, although referred to B-CLL, may have a more general interest, being easily applicable to diseases other than B-CLL with the aim of identifying novel prognostic determinants.The author(s) declare that they have no competing interests.AZ carried out immunophenotypic studies of the original reports and contributed to write the draft of the present manuscript; PS and RC made substantial contribution in the analyses and interpretation of data reported in the original studies and contributed in writing the statistical paragraphs of the present manuscript; MD, RB, MDB and DB performed all the molecular analyses, contributed to the generation of immunophenotypic data of the original reports, and partially contributed to write the present draft; PB, MR and GDP made B-CLL diagnoses and collected clinical data of patients of the original reports; VG coordinated all the original studies, participated in the analyses and interpretation of data and wrote the present manuscript.


In the course of adult hippocampal neurogenesis most regulation takes place during the phase of doublecortin (DCX) expression, either as pro-proliferative effect on precursor cells or as survival-promoting effect on postmitotic cells. We here obtained quantitative data about the proliferative population and the dynamics of postmitotic dendrite development during the period of DCX expression. The question was, whether any indication could be obtained that the initiation of dendrite development is timely bound to the exit from the cell cycle. Alternatively, the temporal course of morphological maturation might be subject to additional regulatory events.We found that (1) 20% of the DCX population were precursor cells in cell cycle, whereas more than 70% were postmitotic, (2) the time span until newborn cells had reached the most mature stage associated with DCX expression varied between 3 days and several weeks, (3) positive or negative regulation of precursor cell proliferation did not alter the pattern and dynamics of dendrite development. Dendrite maturation was largely independent of close contacts to astrocytes.These data imply that dendrite maturation of immature neurons is initiated at varying times after cell cycle exit, is variable in duration, and is controlled independently of the regulation of precursor cell proliferation. We conclude that in addition to the major regulatory events in cell proliferation and selective survival, additional micro-regulatory events influence the course of adult hippocampal neurogenesis. Neuronal development in the adult hippocampus proceeds from the division of a precursor cell to a mature new granule cell ,2. A larDCX is a protein associated with neuronal differentiation that functionally has been linked to cell migration and possible other aspects of maturation, including synaptogenesis . MutatioIn adult hippocampal neurogenesis, the phase of DCX expression coincides with the expression of the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). DCX is transiently expressed during a period that extends from a proliferative progenitor cell stage type-2b/3) to a postmitotic phase with long dendrites . The pha to a posRegulation of adult hippocampal neurogenesis occurs primarily on two stages, both of which might be linked to DCX expression: expansion of the precursor cell pool and selective recruitment of new neurons . We hypoBased on these observations we asked: what is the actual time course of the postmitotic maturation process, most notably with respect to dendrite development, and is it homogenous? Van Praag et al. have described that it takes approximately 7 weeks until by electrophysiological standards the newborn granule cells have become largely indistinguishable from their older neighbors and many studies in several laboratories currently try to link morphological and functional maturation ,26. ZhaoWe wondered if the dynamics of dendrite development were dependent only on the time of the exit from the cell cycle or if any indication could be obtained that the dendritic maturation might be subject to additional regulation. In other words: would non-specific stimuli be acting on a precursor cell stage have consequences on later maturation as well? To address such mechanistic questions it would be necessary to describe the dynamics of early dendritic development during the DCX phase of adult neuronal development. Our aim was to better describe the course of neuronal development in the adult hippocampus encompassing the transition from the expansion phase to functional selection.We first aimed at describing early dendritic development and linking it to mitotic and post-mitotic stages of neuronal development in the adult hippocampus.On tissue sections of the adult hippocampus overviews Fig.  showed tMorphological examination of DCX expressing cells in the SGZ showed a range of morphologies. We categorized DCX-expressing cells into 6 categories according to the presence and the shape of apical dendrites Fig.  and theiDCX is thus expressed during the initial steps of neuronal differentiation, beginning with un-polarized cells with processes parallel to the granule cell layer and ending with a highly polarized cell that extends processes into the molecular layer and shows dendritic branching. All DCX-positive cells in the SGZ and granule cell layer could be categorized according to these criteria. This is in accordance with the idea that during adult hippocampal neurogenesis DCX expression is restricted to the neuronal lineage. Also, morphologically distinguishable interneurons such as basket cells were DCX-negative.We quantified the distribution of DCX-positive cells to these phenotypes and found that more than 65% of the DCX-positive cells belonged to categories E and F  4 hours after the injection of BrdU or the expression of cell cycle-associated protein mki67 with antibody Ki67 . BrdU isThere were no dividing cells in categories E and F, which consequently reflect the postmitotic stages of DCX-expressing cells. We know that the postmitotic stage is also associated with the additional expression of NeuN and CR; CR-positive cells overall showed a morphology similar to DCX-expressing cells in categories E and F . Overalth section (at 40 μm) from 18 mice were processed for TUNEL (TdT-mediated X-dUTP nick end labeling), a feasible way to detect apoptosis. One series was co-stained for DCX-expression, one for CR-expression. TUNEL-positive cells were extremely rare in the hippocampal region. In both series together, only 37 TUNEL-positive cells were detected. These were located either within the SGZ (5 out of 37) or in the granule cell layer proper (32 out of 37). In the DCX-series, 5 out of 17 TUNEL-positive cells showed a DCX immunoreaction , were touched by an astrocytic process 15.8%), or were surrounded by GFAP-positive processes like a basket , we determined the total number of DCX-expressing cells and found almost a doubling , the distribution in the different categories was not influenced. We interpret this result as indicating that at one week after the neurogenic stimulus, progression of DCX-positive cells through the developmental stages highlighted by DCX expression was constant. The number of cells had increased but not their (variable) rate of further development. This might imply that the variability in the duration of DCX expression is not affected by the microenvironmental condition prevalent after experimental seizures. Whereas one study showed an acute proliferative response of GFAP-positive radial cells after experimental seizures , one of We have recently shown that unlike in rats, training the Morris water maze led to a reduction in adult hippocampal neurogenesis in mice, presumably in response to the stress associated with swimming , althougIn the course of adult hippocampal neurogenesis, the newly generated neuronal cells pass through a stage that is characterized by transient DCX expression. In the present study we have added a number of important insights to the knowledge about this crucial phase, which links the precursor cell stage with a postmitotic immature neuronal stage. We show that the phase of increasing dendritic maturation can have variable length but that this pattern, despite its variability, is not influenced by either positive or negative neurogenic stimuli. We propose that these results indicate that although newborn neurons follow a rather fixed pattern of dendritic development and maturation (as captured by the categories A – F), initiation of this development is not only dependent on proliferation per se and exit from the cell cycle, and that regulation of neurogenesis does not overtly include aspects of dendritic maturation.Whereas about 22% of the DCX positive cells were in cell cycle, 68% were showing a co-localization with the strictly postmitotic marker calretinin. This implies that at the given time-point, the remaining 10% of DCX-positive cells are in the progression from a progenitor cell to an early postmitotic stage. The most mature cells (based on dendritic morphology), were always CR-positive. But at one day after BrdU, some CR-positive cells without processes were found in the SGZ. About one third of the DCX-positive cells represent progenitor cells (type-2b and type-3 cells), whereas the remainder are immature postmitotic neurons. This ratio might not be fixed and may be subject to regulation or age-dependent shifts. Within the group of postmitotic cells the time point of CR expression after exit from the cell cycle also differed.We used DCX as a marker here. DCX protein binds to elements of the cytoskeleton, including intermediate filament nestin, which is expressed in precursor cells. In general, DCX expression has been primarily brought into connection with neuronal migration, although other aspects of pathfinding including a role in the growth cones of neurites and in synapse formation have been suggested . Becauseselective survival, e.g. in response to a learning stimulus [A future study will address this hypothesis. However, the experiment is not as straightforward as it might seem because activity-dependent survival is dependent on a critical postmitotic period. So far, no studies in mice have been able to unambiguously demonstrate stimulus ,44. Alsostimulus -52.The largest population of proliferative progenitor cells in the SGZ is DCX-negative. Pro-proliferative stimuli affect DCX-positive and DCX-negative cells . ActivitWe found that the newborn cells leave the close contact to astrocytes, which presumably represents the precursor cell niche, at varying times in the course of development, but separation from the niche is not predictive of the degree of (dendritic) maturation. This might indicate that once the stem cells have produced their differentiating progeny, other factors but the engulfing astrocytic processes become necessary. We and others hypothesize that these factors depend on synaptic input.First synaptic input reaches the newly generated cells of the dentate gyrus on a nestin-positive progenitor cell level and this first input is GABAergic . AmbrogiMany current hypotheses on the activity-dependent regulation of adult hippocampal neurogenesis include a Hebbian mechanism, in which newly generated cells are recruited into the network based on the successful establishment of connectivity early after becoming postmitotic ,58-60. TOur data indicate that the selection and elimination process might take place, when the newly generated cells already have acquired comparatively complex apical dendrites. Because this phase is also associated with TUC4 expression (not shown here) and TUC4 is involved in axon elongation, the selected cells also should have extended their axon to CA3. Nevertheless, it is not yet known, whether the synaptic input that mediates the survival-promoting effect is direct (e.g. from the perforant path), or indirect . In total, synaptic and functional maturation of new neurons takes several weeks and extends beyond the period of DCX and CR expression ,61,62.Synaptic activity is likely to promote cell survival in a similar manner than described for synaptic plasticity. For example, NMDA receptors are likely to be involved in mediating this effect, because blockade of NMDA receptors increased adult neurogenesis -66. MassThis might suggest that the overall dynamics of apical dendrite development were not altered by the seizures. This is not to say that other parameters related to dendritic development not investigated here would have to remain unaffected as well. The morphology, extent and persistence of basal dendrites, for example, is strongly affected by experimental seizures ,67.DCX is thought to be primarily involved in neuronal migration , either As a final note, our data indicate that assessing the number of DCX-positive cells in the dentate gyrus alone as a measure of "adult neurogenesis" might give a misleading impression, because the phase of DCX expression is not only associated with possible cell death (thus reducing quantitative precision) but also encompasses a wide range of qualitative changes.In this report we described that approximately 20% of the DCX expressing population in the adult hippocampus belong to the actively dividing precursor cells, whereas more than 70% of the DCX cells were found to be postmitotic. We found the period of time span until the newborn neurons had reached the most mature stage of dendritic maturation that is still associated with DCX expression varied in length between 3 days and several weeks. Intriguingly, both positive and negative regulators of precursor cell proliferation did not change the pattern and the dynamics of DCX-associated dendrite development. We interpret our findings as indicating that dendrite maturation of adult-generated hippocampal neurons begins at variable time-points after exit from the cell cycle and is variable in duration. Consequently, we hypothesize that the neuronal maturation phase is controlled largely independently from the regulation of precursor cell proliferation and cell cycle exit. This finding has interesting implications for our understanding of how adult hippocampal neurogenesis is regulated in that neuronal maturation does not follow an automated temporal program initiated with fate choice and cell cycle exit but is subject to additional micro-regulatory influences.The experiments were largely done on material from a previously published study . A set oAdditional 18 female C57BL/6 mice (same age as above) were divided into 3 groups: seizure (N = 6), running (N = 6) and controls (N = 6). Seizure animals received a single intraperitoneal application of 30 mg/kg kainic acid  in 0.1 M phosphate buffered saline (PBS) on the day before BrdU-injections (Day 0), and only those displaying continuous convulsive seizure activity were used in these experiments. The "Runner" group was housed with 2–3 animals per cage that was equipped with a running wheel. During the first 7 days of the experiment all animals received one daily injection of BrdU. Tissue from this experiment will be used in an unrelated study. Data on dendritic morphology, etc., were exclusively generated for the present study.To analyze the spatial relationship between DCX-positive cells and astrocytes we used 3 female transgenic mice expressing enhanced green fluorescent protein EGFP under the promoter for glial fibrillary acidic protein (GFAP). The animals were kindly provided by Helmut Kettenmann, Berlin  and wereFor the water maze experiment, 21 female Nestin-GFP reporter mice  . The brains were removed from the skull, were kept in the fixative for 24 h and then transferred into 30% sucrose, where they remained until equilibrium. Forty μm coronal sections were cut from a dry ice-cooled copper block on a sliding microtome . The sections were stored at -20°C in cryoprotectant containing 25% ethylene glycol and 25% glycerin in 0.05 M phosphate buffer. Sections were stained with free floating immunohistochemistry and pretreated for BrdU-detection by incubation in 2 N HCl for 30 min at 37°C followed by washing in 0.1 M borate buffer (pH 8.5) for 10 min.Brain tissue for the TUNEL assay (see below) was rapidly frozen in liquid nitrogen and cut on a cryostat at 15 μm. Sections were mounted onto Superfrost coated glass slides, air-dried, post-fixed with 4% paraformaldehyde (20 min) and thoroughly washed in phosphate buffered saline (PBS).To determine the number of BrdU-labeled and DCX-expressing cells we used the peroxidase method  with biotinylated donkey anti-rat and anti-rabbit antibodies  and nickel-intensified diaminobenzidine as chromogen. Immunofluorescent triple labeling was done as described earlier .The primary antibodies used in our study were monoclonal rat anti-BrdU , monoclonal mouse anti-NeuN , monoclonal mouse anti-Calretinin , rabbit anti-S100β , goat anti-Doublecortin , polyclonal rabbit anti-Ki67 ; polyclonal rabbit anti-GFP ; monoclonal mouse anti-phospho histone H3 .As fluorescent secondary antibodies we used anti-rat or anti-mouse Rhodamine-X, anti-mouse or anti-rabbit FITC, anti-mouse, anti-rabbit or anti-goat Cy5 . Fluorescent sections were mounted in polyvinyl alcohol with diazabicyclo-octane (DABCO) as anti-fading agent.We confirmed the specificity of our antibody against doublecortin, on which the results of the present study are based, using a western blot of homogenate from mouse hippocampus (not shown). The antibody showed the expected band below 50 kD. This reaction was completely blocked with the DCX peptide.Mouse hippocampi were lysed with RIPA buffer  and homogenized with an ultrasonic homogenizer (setting 40 Hz) for 60 seconds and cellular debris was removed by 15 minutes centrifugation at 14 000 rpm. Total protein content was quantified using a Bio-Rad protein reagent (Bio-Rad Laboratories GmbH. München). Aliquots of 40 μg of protein were separated by electrophoresis on 12% SDS-polyacrylamide gels by the Laemmli method  and tranth section (240 μm apart) were stained for BrdU- and for DCX with the peroxidase method. Positive cells were counted using a 40 × objective (Leica) throughout the rostro-caudal extent of the granule cell layer. As described previously, the optical dissector method was modified in that cells appearing sharp in the uppermost focal plane were not counted [From all animals series of every 6 counted . ResultiTo categorize DCX-positive cells according to the 6 morphological grouping criteria described in this study, 100 DCX-labeled cells were analyzed per animal.One-in-twelve series of sections from animals of each group were triple-labeled for BrdU, GFP and DCX or double-labeled for BrdU and DCX. Fluorescent signals were detected using a spectral confocal microscope (Leica TCS SP2). All analyses were performed in sequential scanning mode to rule out cross-bleeding between channels. Double-labeling was confirmed by three-dimensional reconstructions of z-series covering the entire nucleus (or cell) in question. Three-dimensional reconstructions were performed with Volocity 2.0 software (Improvision).From each animal and for every combination of phenotypes 50 BrdU-positive cells within the granule cell layer were analyzed at the confocal microscope for co-expression of the different markers. Relative numbers were related to the absolute counts of BrdU-positive cells per granule cell layer to yield the absolute numbers of BrdU-marked cells per phenotype. Images were processed with Adobe Photoshop 7.0, and only general contrast enhancements and color level adjustments were carried out.The TUNEL  method visualizes apoptotic cells by detecting free 3' ends of DNA strand breaks . We usedAll numerical analyses were performed using Statview 5.0.1. For all comparisons ANOVA was performed followed by Fisher's post hoc test, if appropriate. Differences were considered statistically significant at p < 0.05. The post hoc analyses were additionally run with Students-Newman-Keuls test, which did not yield differing results (not shown).TP, SJ and GK designed the study; TP, DE, BS, FK, MB, BR, GKr, and GR, designed and carried out the individual experiments and acquired the data; TP, SJ, BS, GR, GKr, and GK analyzed the findings in the light of the present hypothesis; TP and GK wrote the manuscript with support from the other co-authors. All authors read and approved the final manuscript.


Analysis of 2,086 conserved sequence blocks (CSBs), identified from 135 characterized enhancers, reveals most CSBs consist of shorter overlapping/adjacent elements that are either enhancer type-specific or common to enhancers with divergent regulatory behaviors. Our findings suggest that enhancers employ overlapping repertoires of highly conserved core elements.A systematic approach is described for analysis of evolutionarily conserved  Enhancers, which usually span 0.5 to 1.0 kb, contain clusters of transcription factor DNA-binding sites  enhancers that drive expression in the same embryonic neuroblasts, yet comparisons of these enhancers reveal that they have few sequences in common [Tissue-specific coordinate gene expression requires multiple inputs that involve dynamic interactions between sequence specific DNA-binding transcription factors and their target DNAs. The enhancer or iewed by ). DNA sen common ,5.cis-regulatory regions reveals that many contain multi-species conserved sequences  of collective divergence [Drosophila Krüppel central domain enhancer [Drosophila characterized enhancers reveal that well over half of the sequences do not correspond to known DNA-binding sites and, as yet, have no assigned function(s) (this paper).Comparative genomic analysis of orthologous iewed by -8). Closvergence  , within enhancer CSBs that are also present in CSBs from other enhancers with either related or divergent functions. There is no limit to the number of enhancer CSBs examined by this approach, which allows one to build large cDT-libraries. Due to their different copy numbers, positions and/or orientations within the different enhancers, the conserved short sequence elements may otherwise go unnoticed by more conventional DNA alignment programs. Because this approach does not rely on any previously described transcription factor consensus DNA-binding site information or any other predicted motif or the presence of overrepresented sequences, cis-Decoder analysis affords an unbiased 'evo-centric' view of shared single or multiple sequence homologies between different enhancers. The cDT-libraries and cis-Decoder alignment tools enable one to differentiate between functionally different enhancers before any experimental expression data have been collected. cis-Decoder analysis reveals that most CSBs have a modular structure made up of two classes of interlocking sequence elements: those that are conserved only in other enhancers that regulate overlapping expression patterns; and more common conserved sequence elements that are part of divergently regulated enhancers.In order to initiate the functional dissection of novel CSBs and to gain a better understanding of their substructure, we have developed a multi-step protocol and accompanying computer algorithms  and Drosophila snail genes, which contain closely associated neural and mesodermal enhancers, were selected to highlight cis-Decoder's ability to differentiate between enhancers with different regulatory functions. We show how a cDT-library generated from both mammalian and Drosophila enhancer CSBs can be used to identify enhancer type-specific elements that have been conserved during the evolutionary diversification of metazoans. Finally, we show how cis-Decoder analysis can be used to examine novel putative enhancer regions.To demonstrate the efficacy of cis-regulatory sequences included 14 neural and 21 mesodermal enhancers whose regulatory behaviors have been characterized in developing mouse embryos. A full list of enhancers used in this study and the references describing their embryonic expression patterns is given in Table EvoPrints included orthologs from placental mammals  or also included the opossum; these species afford enough additive divergence (≥200 My) to resolve most enhancer MCSs [EvoPrints. Except when EvoDifference profiles [EvoPrints.Our analysis of mammalian cer MCSs . When poprofiles  revealedEvoPrint-Parser program, both forward and reverse-complement sequences of each enhancer CSB of 6 bp or greater were extracted, named and consecutively numbered. Based on their enhancer regulatory expression pattern, CSBs were grouped into two different CSB-libraries, neural and mesodermal  segmental enhancer [cis-Decoder website [For g Tables  and 2: ne REDfly , while oy [snail , scratchnhancers , and latcer ey12 , which cstripe 1  and evenstripe 1  enhancernhancers ,20. The s [snail  and tinm [tinman , and latpression  and meso reduced . The colenhancer ,24 and 9enhancer . Complet website .cis-Decoder, that allow identification of 6 bp or greater perfect match identities, called cDTs, within two or more CSBs from either similar or divergent enhancers. The cDTs, which range in size from 6 to 14 bp with an average of 7 or 8 bp, are organized into cDT-libraries that identify sequence elements within CSBs of the same CSB-library. In addition, common cDT-libraries that represent sequence elements aligning to CSBs of two or more different CSB-libraries were also organized.As an initial step toward understanding the nature of the CSB substructure, we have developed a set of DNA sequence alignment tools, known collectively as CSB-aligner program, yielded 336 neural specific and 60 neural-enriched cDTs and analysis of the mammalian mesodermal CSBs yielded 258 mesodermal specific and 55 mesodermal enriched cDTs , 284 segmental enhancer specific cDTs and an additional 451 cDTs found in neural and segmental enhancers but not part of mesodermal CSBs  were identified along with 104 additional cDTs enriched in mesodermal enhancers but also found at a lower frequency among neural and/or segmental enhancer CSBs. A common cDT-library was also generated that contains 993 cDTs that represent common sequence elements found in CSBs of both neural and mesodermal enhancers.Mammalian CSB alignments, using the cDT-libraries from the combined alignments of mammalian and fly CSBs  aligns with CSBs in the vertebrate Math-1 [Drosophila deadpan [cDT-libraries and alignment tools are available at the cis-Decoder website.To search for enhancer sequence element conservation between taxa, we generated neural and mesodermal Bs Table  and manye Math-1  and Dros deadpan  early CNcDT-libraries are dependent on the enhancers used to identify them. As additional CSBs are included in the cDT-library construction, certain cDTs may be re-designated. For example, some that are currently considered neural specific will be discovered to be neural enriched, and others that are part of enriched libraries may be reassigned to common cDT-libraries.The constituent sequence elements of the different cDT is present in at least two or more enhancers, most are not found as repeated sequences in any of the enhancers. In addition, one of the principle observations of our analysis is that enhancers of similarly regulated genes share different combinatorial sets of elements that are enhancer-type specific (see below).Although each mammalian and fly cDTs that are either shared by CSBs from divergent enhancer types or found only in CSBs from enhancers with related regulatory functions. For example, the 37 bp neural mastermind #10 CSB (TATTATTACTATATACAATATGGCATATTATTATTAC) contains a 9 bp sequence (first underlined sequence) also found in the 20 bp #8 CSB from the dpp mesodermal enhancer [#33 CSB from the neural enhancer region of nerfin-1  transcription factors, the E-box motif CAGCTG . It has been previously shown that different E-boxes bind different bHLH transcription factors to regulate different neural target genes [cDT-libraries, greater than 50% of the cDTs in all of the libraries, both mammalian and fly, represent novel sequences whose function(s) are currently unknown. The fact that there exists such a high percentage of novel sequences within these highly conserved sequences indicates that the identity, function and/or the combinatorial events that regulate enhancer behavior are as yet unknown.The analysis of both the mammalian and fly common iewed by ) is preset genes . Althougcis-Decoder analysis increases as more enhancers and/or enhancer types are included in the CSB and cDT alignments, our analysis of mammalian enhancers found that many shared sequence elements can be identified among related enhancers when as few as two different enhancer groups are used to generate specific cDT-libraries. This is a particularly useful feature of cis-Decoder, especially when studying a biological process or developmental event where relatively little is known about the participating genes and their controlling enhancers. To demonstrate the ability of cis-Decoder to analyze relatively small subsets of enhancers, we show how cDT-libraries generated from 14 neural and 21 mesodermal mammalian enhancers can be used to distinguish between the neural and mesodermal enhancers that regulate embryonic expression of Dll1.Although the resolution of cis-regulatory region, located 5' to its transcribed sequence, has been shown to contain distinct enhancers that direct gene expression in these different tissues [Dll1 encodes a Notch ligand that is essential for cell-cell signaling events that regulate multiple developmental events (reviewed by ). Studie tissues . These s tissues . The msdEvoPrint of the Dll1 cis-regulatory region reveals clustered CSBs in each of the enhancer regions , human, rhesus monkey, cow, rat, opossum and Xenopus tropicalis orthologs, representing over approximately 240 My of collective evolutionary divergence. EvoPrint-parser CSB extraction of the EvoPrint generated a total of 35 CSBs of 6 bp or longer, representing 83% of the total MCS. A cDT-scan of the four Dll1 enhancer regions using the mammalian neural and mesodermal specific cDT-libraries accurately differentiates between the neural and mesodermal enhancers  align with three or more CSBs from different enhancers, indicating that, even if Dll1 had been excluded from the analysis that generated the specific cDT-libraries, there would still be extensive coverage of the Dll1 CSBs by type-specific cDTs. All but eight of the CSBs contain elements that align with one or more neural or mesodermal specific cDTs. The H-I and H-II early CNS enhancers exhibited 64% and 43% coverage, respectively, by neural specific cDTs. The CSBs of the two mesodermal enhancers, msd and msd-II, exhibited 48% and 56% coverage, respectively, by one or more mesodermal specific cDTs. When common cDTs, shared by mesodermal and neural enhancers, were taken into account, coverage of all four enhancers was 81% (data not shown).An s Figure . Here, EcDT-cataloger analysis of aligning cDTs with H-I and H-II early CNS enhancers revealed that the H-I enhancer shares a remarkable 9 different sequence elements with the Wnt-1 early CNS neural plate enhancer CSBs [p hindbrain-spinal cord enhancer CSBs [p enhancer CSBs reveal that the orientation and order of the shared cDTs are unique for each of the enhancers (data not shown). The H-I and H-II enhancer CSBs also share the 7 bp sequence element GCTCCCC, and H-I has a repeat sequence element (AGTTAAA) that is present in two of its CSBs (#11 and #13). The conserved AGTTAAA repeat is also part of a CSB in Phox2b enhancer [cDT-cataloger analysis of the mesodermal enhancer cDT hits  in the CSB alignments, the total number of neural specific cDTs increased to 873 compared to 336 mammalian and 322 Drosophila neural specific cDTs . For ex in mice  and its osophila . Given trs Table . By incls Figure , suggestcis-Decoder to differentiate between Drosophila neural and mesodermal enhancers, we show an analysis of the snail upstream cis-regulatory region. The enhancers that regulate snail's dynamic embryonic expression have been mapped to a 2,974 bp upstream DNA fragment [EvoPrint of this sequence reveals that each of the restriction fragments that contain the different enhancer activities  harbor clusters of highly conserved CSBs  is approximately 160 My, suggesting that many, if not all, of the identified CSBs are likely to be genus invariant and that each base-pair within a CSB has been evolutionarily challenged.To demonstrate the ability of fragment . An EvoPs Figure . The comsnail upstream CSBs that are present in CSBs of other functionally related or unrelated enhancers, we carried out a cDT-scan of the snail EvoPrint using the neural, segmental and mesodermal specific cDTs and the enriched cDT-libraries  contains a consensus core DNA-binding sequence (underlined) for Antennapedia class homeodomain factors [Within the presumptive mesodermal enhancer CSBs, 11 s Figure . Like th factors  (reviewe factors ).snail early PNS enhancer region, 5 of the 7 CSBs aligned with a total of 15 different cDTs that cover 69% of the total PNS CSB sequence . cis-Decoder recognizes highly conserved Abdominal-B, HOX, Hunchback, Kruppel and Tramtrack binding sites, as well as additional uncharacterized sites, as being shared by hairy stripe 1 enhancer and other segmentation enhancers.As part of our study of Krüppel central domain enhancer [In vivo experiments reveal that these interlocking sites are functionally important [Krüppel enhancer but not all are found in CSBs (data not shown).Previous studies have demonstrated that certain enhancers, particularly those controlling the dynamic expression of developmental genes, contain clusters of DNA-binding site motifs for specific transcription factors  is present two additional times within the intra-CSB sequences  is conserved in all Drosophila species except D. ananassae and the 3' inter-block repeat is absent in six of the ten species used to generate the EvoPrint (data not shown).The s Figure . Interescis-Decoder methodology is the comparative analysis of different enhancer regions. To test cis-Decoder's efficacy in characterizing putative cis-regulatory regions that were not included in the preparation of the cDT-libraries, we have examined a number of genes both in Drosophila and vertebrates using EvoPrinter and cDT-library scans. Our analysis reveals that putative enhancer regions associated with CNS-expressed genes align with a higher proportion of neural-specific cDTs than with mesodermal-specific cDTs. For example, cis-Decoder analysis of the immediate upstream regions from Drosophila E(spl) region transcript mβ (HLHmβ) [cDTs of their proximal cis-regulatory region CSBs. Figure cis-Decoder analysis of HLHmβ, while our analysis of TIP39 is presented in Additional data file 3.One major use of the  (HLHmβ)  and of t (HLHmβ) -53 reveaHLHmβ expression is activated in the ventral neurogenic ectoderm immediately prior to neuroblast delamination [HLHmβ enhancer region [EvoPrint analysis of the 883 bp enhancer region , the focus of activity of proneural transcription factors, and two others, TTTCCCA and TCCCAC, align with the consensus Su(H) binding site.During embryonic development, mination ,54 and er region  are exprr region ). Our Evn Figure  revealedT Figure . Both prcDTs of 7 bases or greater, we have found that it is not unusual for 80% of CSBs associated with neural expressed genes to align with neural-specific cDTs versus only 20% of the CSBs in the same putative enhancer regions aligning with mesodermal-specific cDTs even when 6 base long cDTs are included in the analysis (data not shown). As the size and specificity of these libraries grow, their use as predictors of enhancer function will most likely increase as well.Although higher specificity is obtained by alignment with cDT-library scans, we generated negative control CSB-libraries for alignment to cDTs. These datasets, both Drosophila and mammalian, consisted of conserved sequence blocks within exons of genes that are not predominantly expressed in the CNS (data not shown). For this analysis we use the percent coverage of CSBs by cDTs, as used above for the analysis of Dll1 enhancers in which we counted the percent of the bases in the CSBs that aligned with cDTs. Whereas Drosophila and mammalian neural-specific cDTs, including hexamers, cover approximately 56% and 70%, respectively, of CSBs from neural enhancers, alignment with control CSBs was 20% or less. Again, when the alignment was repeated with cDTs of 7 bp or greater the CSB coverage of neural sequence was 5-fold greater than that observed with the control datasets. Taken together, our cDT alignments demonstrate their utility in identifying enhancer type-specific conserved sequence elements.As an additional assessment of the specificity of cis-Decoder method was also carried out by examining the contribution that each enhancer made to the cDT-libraries. As one adds new enhancer CSBs to a specific library, the number of cDTs increases, such that alignment coverage of enhancer type-specific CSBs also increases. We illustrate the contribution of each enhancer to the specific cDT-libraries in our study . Overall, for Drosophila enhancers, prior to their inclusion in a library, on average 41% of the conserved nucleotides of enhancers align with the tissue specific cDT-library appropriate for that enhancer, while after inclusion in a library, 65% of the conserved nucleotides align. For example, addition of the bearded proneural enhancer [Drosophila neural-specific CSB library resulted in 26 new neural-specific cDTs that were shared with at least one other neural enhancer. Prior to its inclusion, coverage of the bearded CSBs by alignment of neural-specific cDTs was 43%, while after its inclusion in the cDT-library preparation the alignment coverage of its CSBs increased to 67%. Addition of new enhancers to the out-group, used to remove common cDTs from a specific library, also enhances the specificity of the type-specific library and frequently shifts cDTs from specific to enriched libraries. Taken together, increased specificity of an enhancer-type cDT-library can be achieved either by including new similarly regulated enhancers in the generation of the cDT-library or increasing the number of out-group CSBs used to remove non-specific cDTs. Ideally, both approaches should be pursued to increase the depth and resolution of a particular cDT-library.Evaluation of the enhancer , consistcis-Decoder analysis dissects functionally important DNA is greatly enhanced. Most of the 2,086 CSBs identified in this study have undergone negative selection during more than 160 My of collective evolutionary divergence. Alignment of hundreds of CSBs from both similarly regulating enhancers and functionally different enhancers assures that conserved cis-regulatory elements shared by as few as two enhancers are identified and included in the analyses. Our cDT-scans show that most CSBs have a modular organization made up of smaller overlapping/interlocking sequence elements that align with CSBs of other enhancers. A typical CSB is made up of both enhancer type-specific sequence elements and common elements that are found in enhancers with different regulatory functions and, surprisingly, more than half of all of the shared CSB sequence elements do not correspond to know transcription factor DNA-binding sites and, as of yet, are functionally novel.This study describes a systematic approach for the identification and comparative analysis of highly conserved DNA sequences within enhancers. Because our approach focuses solely on conserved sequences, the probability that cDT-library scans of EvoPrinted cis-regulatory DNA reveal that it is possible to differentiate between functionally different enhancer types before any experimental/expression data are known. For example, cDT-library scans of the mammalian Dll1 or Drosophila snail cis-regulatory DNA sequences accurately differentiate between neural and mesodermal enhancers , and, optionally, the opossum, detects CSBs that have been maintained for over 200 My of collective divergence. The EvoPrinter and EvoDifference print analysis algorithms and companion protocols are described [EvoPrinter tutorial website.The first step in the oPrinter . Unlike EvoPrint-parser is a JavaScript program that automatically extracts and generates reverse-complement sequence and then annotates and lists in their 5' to 3' order CSBs that are 6 bp or longer from a known or putative enhancer region. Tissue-specific enhancer CSB-libraries can then be generated by assembling CSBs from enhancers of known function .The CSB-aligner is a Java applet that allows one to identify short sequence elements shared between different CSBs. To generate a CSB-alignment, parsed CSBs from multiple enhancer regions are placed in the upper window of the CSB-aligner applet. Then, forward direction CSBs from one or more enhancers are placed in the lower window of the CSB-aligner. A box associated with the lower window of the CSB-aligner allows for the naming of the CSBs introduced into the lower box and selection of the minimum aligned length . Output length of the alignments produced by CSB-aligner can be selected .CSB-aligner consists of the CSBs that were input into the lower window aligned with the CSBs that were introduced into the upper window. The CSB-aligner does not record CSB self-alignments. A second output window, the results table, is a list of the aligned matches along with their positions. Each of the output columns of the results table can be sorted by selecting the column header of the column to be sorted. Contents of results tables can be copy-pasted into Microsoft Word.Output of the CSB-alignment program has functioned successfully with the introduction of thousands of CSBs in both windows. The following CSB-libraries were created from EvoPrints of enhancers listed in Table Drosophila neural, Drosophila mesodermal and Drosophila segmental.The CSB-alignment can be saved as an HTML file. Saving the HTML file allows copy pasting from the saved file into Microsoft Word and, once in Word, the file can be reformatted and saved or printed as the original readout. The cDT is a short sequence element of 6 bp or greater that is a perfect match to sequences within CSBs that are present in two or more enhancers. A cDT-library represents a collection of cDTs that are shared by the various enhancers examined. Two types of cDT-libraries have been generated in this study. First, a 'tissue-specific library' contains cDTs that are shared by a group of enhancers that regulate similar expression patterns but are absent from a second set of enhancers that direct expression in tissues outside of the first group. Second, a 'common cDT-library' contains cDTs that were shared between sets of enhancers of divergently regulated genes. A subset of common libraries included 'enriched' libraries that had a three-fold greater representation from one enhancer type  than from a second type .A CSB-aligner. Making enhancer-type specific libraries requires two different CSB-libraries generated from functionally different enhancers, a library from the tissue of interest , and a second library that serves as an 'out-group' . For the generation of a neural cDT-library, neural CSBs in both forward and reverse directions were copy-pasted into both upper and lower windows of CSB-aligner. The resulting cDTs from this alignment are listed in the 'Result of CSB alignment table' of the CSB-aligner output, in the column titled 'Motif.' Since this cDT list contains multiple copies of different cDTs, the extra copies are removed using the Java applet Puzzamatic 1.0 [cDT list that contains all unique cDTs is then alphabetized and sorted by size also using Puzzamatic 1.0. The cDTs, constituting a raw neural cDT-library, were then copy/pasted into a Microsoft Word document. A second CSB-alignment is then performed with the neural CSBs in the top window of CSB-aligner, and mesodermal CSBs in the lower window. The cDTs from this alignment were freed of extra copies as above. These cDTs constituted an unedited common neural/mesodermal cDT-library. The unedited neural and common cDT-libraries are combined and cDTs common to the two libraries  are removed using the JavaScript program cDT-cleaner [cDTs were curated from the unedited shared cDT-library.All libraries were generated from readouts of the atic 1.0 , a freew-cleaner , thus leDrosophila, segmental, neural (treating CNS and PNS specific enhancers together), and mesodermal specific cDT-libraries were generated. The out-group for neural and segmental cDT-libraries was the mesodermal CSB-library, and the out-group for the mesodermal cDT-library was neural CSBs. For mammals, neural and mesodermal cDT-libraries were generated. All cDT-libraries are listed in Table For cDT-scanner is to determine the relationship between any enhancer and any other group of MCSs used to generate the CSB libraries. cDT-scanner aligns the cDTs contained within various cDT-libraries with CSBs within an EvoPrint. cDT-scanner is a Java applet that uses a variant of the cis-Decoder aligner; it looks for only perfect matches between cDTs and CSB sequences. Alignment of cDTs using cDT-scanner is accomplished by first pasting a cDT-library in the upper window of cDT-scanner and then pasting the EvoPrint or CSBs to which they are to be aligned in the lower window. The output of cDT-scanner consists of perfect matches of cDTs aligned under the input CSBs. Since each library consists of cDTs shared by different enhancers, cDT-scanner portrays the shared elements within each CSB. A cDT-scanner alignment should be saved; information from saved files can be copy-pasted into Microsoft Word without loss of formatting features. For details on how to format cDT-alignments, see the website. A second output window for the cDT-scanner, a results table, is a list of the aligned matches along with their positions. Selecting the output column header sorts the results under that header. Contents of results tables can then be copy-pasted into Microsoft Word.The function of Full-enhancer scanner' is a Java applet that identifies additional repeated cDT or CSB sequences within less conserved sequences flanking CSBs of enhancers. For this alignment, cDTs or CSBs present within an enhancer can be curated from the output of cDT-scanner termed 'Results from cDT-scan.' Curate both forward and reverse/complement sequences and paste into the upper window of Full-enhancer scanner. The EvoPrinted enhancer should be copy-pasted into the lower window. The program aligns to both conserved and non-conserved sequences of the EvoPrint.The 'cDT-cataloger uses a variant of the CSB-aligner; it records only perfect matches between CSBs and cDTs of a specified size. The output lists those CSBs containing perfect sequence matches to the cDTs, and can be used to identify enhancers and count the number of times each cDT aligns with any CSB-library. Cataloguing is accomplished by copy-pasting the CSB-libraries (both forward and reverse directions) into the upper window of the cDT-cataloger and the selected cDTs of a single uniform size in the lower window. The size of the cDT(s) must be entered into the window provided.cDT-cataloger analysis of the murine Delta-like 1 Homology-II and msd-II enhancers supplemental to Figure cis-Decoder analysis of the Drosophila hairy stripe 1 enhancer. Additional data file cis-Decoder analysis of the human TIP39 5' proximal promoter. Additional data file Drosophila and mammalian enhancer to the specific cDT-libraries generated in this study.The following additional data are available with the online version of this paper. Additional data file cDT-cataloger analysis of the murine Delta-like 1 Homology-II and msd-II enhancers supplemental to Figure 4Click here for filecis-Decoder analysis of the Drosophila hairy stripe 1 enhancerClick here for filecis-Decoder analysis of the human TIP39 5' proximal promoterClick here for fileDrosophila and mammalian enhancer to the specific cDT-libraries generated in this studyContribution of each Click here for file