Vertebrate embryonic cells recognize self from non-self, thus restricting repulsion at tissue boundaries, through a combination of multiple ephrins and Eph receptors, simply based on binding selectivity and asymmetric expression. Ephrins and Eph receptors are involved in the establishment of vertebrate tissue boundaries. The complexity of the system is puzzling, however in many instances, tissues express multiple ephrins and Ephs on both sides of the boundary, a situation that should in principle cause repulsion between cells within each tissue. Although co-expression of ephrins and Eph receptors is widespread in embryonic tissues, neurons, and cancer cells, it is still unresolved how the respective signals are integrated into a coherent output. We present a simple explanation for the confinement of repulsion to the tissue interface: Using the dorsal ectoderm–mesoderm boundary of the Xenopus embryo as a model, we identify selective functional interactions between ephrin–Eph pairs that are expressed in partial complementary patterns. The combined repulsive signals add up to be strongest across the boundary, where they reach sufficient intensity to trigger cell detachments. The process can be largely explained using a simple model based exclusively on relative ephrin and Eph concentrations and binding affinities. We generalize these findings for the ventral ectoderm–mesoderm boundary and the notochord boundary, both of which appear to function on the same principles. These results provide a paradigm for how developmental systems may integrate multiple cues to generate discrete local outcomes. How embryonic tissues separate from each other to shape the developing organism is a fundamental question in developmental biology. In vertebrates, this process relies on local repulsive reactions specifically generated at contacts between cells of different types. These reactions are triggered by typical repulsive cell surface cues, the ephrin ligands, and Eph receptors. However, the expression of multiple ephrins and the Eph receptors by each cell type represents a puzzle: Why is repulsion observed only at the tissue interface and not within the tissue itself? By studying three cases of separation in the early amphibian embryo, we uncover a surprisingly simple logic underlying this phenomenon, which can be explained by the selectivity of ligand–receptor interactions and by their asymmetric distribution. The system is set such that, despite generalized interactions throughout the tissues, it is only at contacts between different cell types that the overall repulsive output is sufficiently strong to overcome cell–cell adhesion. Our study may serve as paradigm for how systematic dissection of complex cellular systems can reduce them to simple laws and make them intelligible. In vertebrates, ephrins and Eph receptors have emerged as major players in the formation of cleft-like tissue boundaries. They control segmentation of rhombomeres Understanding how the global output is determined under in vivo conditions has thus remained a daunting challenge. An example of where the integration of multiple co-expressed Eph receptors and ephrins can be tested is the ectoderm/mesoderm boundary in the early Xenopus embryo. We have demonstrated that ephrins and Ephs act directly at the tissue interface, where they generate cycles of attachments and detachments through transient activation of Rho GTPases To address the issue of repulsion restriction, we conducted a comprehensive characterization of the ephrin–Eph system in the early gastrula. We first compared quantitatively the transcripts of all ephrins and Ephs in the dorsal ectoderm and in the mesoderm. Both tissues expressed multiple ephrins and Ephs. Although both A and B types are involved at the ectoderm–mesoderm boundary . Thus, eAlthough ephrin/Eph depletion severely disrupts the endogenous ectoderm–mesoderm boundary , the embAfter having established that each ephrin and Eph subtype is required, we asked next whether a given subtype could be replaced by another member of the family. An ephrin or Eph was depleted, and rescue was attempted by mRNA injection , or by dThe specific requirement for all ephrinB subtypes and their receptors could be due to specific differences in downstream signaling and hence to different roles during tissue separation. Alternatively, signaling could be uniform and additive, but depend on specific receptor–ligand interactions. To determine whether Eph receptor specificity resided in their cytoplasmic tail or in their extracellular domains, we constructed EphA4/B4 chimeras, where the respective cytoplasmic domains were swapped, which we tested for the ability to substitute for endogenous EphA4 or EphB4 proteins. We verified that these chimeric constructs were properly expressed at the cell surface  and actiIf indeed specific ephrin–Eph pairs formed preferentially at the boundary, we predicted that ectopic addition of ephrins normally enriched in the mesoderm should induce artificial separation of two ectoderm explants. This prediction was fully verified. Indeed, ectoderm explants could be induced to repel each other by treatment with soluble Fc fragments for mesoderm-enriched ephrinB2 but not ephrinB1 . SimilarThe sensitivity of ectoderm and mesoderm to respond to a subset of ephrin/Eph fragments implied that each of these two tissues must specifically express respective partners. We used the induction of ectopic separation as a functional assay to identify these endogenous receptors. Explants depleted of single ephrins or Ephs were tested for their ability to separate upon incubation with an Fc fragment. Among potential candidate receptors for ephrinB2 in the ectoderm, EphB4 depletion strongly inhibited ephrinB2-Fc–induced separation, whereas EphB2 depletion had a weaker effect . The abiWe directly assessed functional selectivity at the level of Eph activation . After tTaken together, our results show that each Eph receptor is selective for one or at most two ephrins. These results support the hypothesis that tissue separation is driven by asymmetric expression of specific receptor–ligand pairs. Most of the ephrin/Eph pairs identified functionally show indeed partially complementary expression . This isOur results suggested that tissue separation may be simply explained by the complementary expression of selective ephrin/Eph pairs, which would generate an excess of repulsive signal at the boundary . NeverthWe tested the specificity of the process by attempting the rescue with other ephrins or Ephs. EphrinB2, which is another good partner for EphA4, could substitute for ephrinB3 . No rescAlthough EphA4 and B4 cytoplasmic tails appear interchangeable , we alsoImmunostaining of sections from wild-type gastrulae with an antibody recognizing a conserved phosphorylated site present in all EphBs demonstrated that Eph signaling was indeed activated in both ectoderm and mesoderm, but was significantly stronger at the boundary . A similThe typical mechanical output of Ephrin/Eph signaling in repulsive behavior involves myosin-based contraction Significant Ephrin–Eph signaling also takes place within each of the two tissues . To accoThis model predicted that increasing cadherin levels or decreasing contractility should inhibit detachments between ectoderm and mesoderm cells, while on the contrary decreasing cell adhesion or increasing Eph signaling should lead to visible detachments between mesoderm cells. This hypothesis was consistent with the observation that ectoderm–mesoderm separation was impaired upon cadherin overexpression, which could be rescued by boosting Eph signaling across the tissue interface by incubation with soluble ephrin Fc fragments . Note thTo explore the behavior at individual homotypic and heterotypic cell–cell contacts, we juxtaposed single cells obtained by dissociation of early gastrula tissues –S7. Our Because we expected mild repulsion between mesoderm cells, we predicted that detachments should become more prominent if adhesion would be experimentally decreased. We subjected mesoderm cells to a mild cadherin depletion  and observed that contacts were now much less stable and cells displayed the typical repulsive behavior normally observed at contacts with ectoderm cells . We alsoOur results suggested that separation could be explained by the asymmetric expression of a subset of specific ephrin–Eph pairs, which resulted in widespread ephrin–Eph signaling, but with an altogether higher output across the boundary. Although the concept is intuitively coherent, the actual system is complex, with many ephrins and Ephs, including pairs that could interact extensively within one tissue . To better estimate the validity of this model, we simulated it by computing the contribution of all the various ephrin–Eph interactions established at homotypic contacts within the tissues and heterotypic contacts between tissues . We consHowever, this simple model where receptor activation depends on its own concentration, the concentrations of its various potential ligands, and the corresponding affinities turned out to describe with surprising accuracy the situation at this boundary. The signal output turned out indeed to be clearly highest at the tissue boundary compared to ectoderm–ectoderm and mesoderm–mesoderm contacts . The sysWe conclude that a system of partially complementary and semiselective ephrin–Eph pairs is indeed sufficient to explain ectoderm–mesoderm separation. The robustness of the simulation provides strong support for the general principles of this model. Although multiple additional factors are likely to impact on the apparent concentrations and affinities, on the response curves of Eph activation, and more downstream on the contractile activity of boundary cells, such factors may not affect significantly the final global pattern. The reason for this robustness appears to be the very limited set of pairs that can effectively interact and thus influence the system, and the fact that three out of five of these pairs are asymmetrically expressed . The simulation indicated that, for these five pairs, differences up to 10-fold in apparent affinities would change the relative strength of the outputs, but the signal remained strongest at the boundary under most conditions. The few aspects of our experimental data that were not well simulated are discussed in We wondered whether the principles found for the early ectoderm–mesoderm boundary would similarly apply to other boundaries. Ephrin and Eph expression is indeed widespread in the gastrula embryo, and two other important boundaries form during this phase. A ventral boundary appears at midgastrula to separate the ectoderm for the ventral mesoderm , which iThese observations suggested that tissues that became separated by boundaries expressed some sort of “modules” characterized in particular in all cases by complementary ephrinB3–EphA4 expression, but had some flexibility for other components. We then tested the role of key players, at both boundaries. In the case of the ventral boundary, we used the classical separation assay, attempting both loss- and gain-of-function experiments . All theIn gain-of-function experiments, both mesoderm-enriched ephrinB1 and B2 could induce robust separation between two ectoderm explants when added as soluble Fc fragments , an effeThe role of ephrins and Ephs on the notochord boundary was examined by targeted depletion in a restricted region of the embryo This study reconciles several previous puzzling observations and provides a coherent description of boundary formation based on selective repulsion between two cell populations. In particular, it explains how in tissues with widespread expression of multiple ephrin ligands and Eph receptors, cell–cell repulsion due to receptor–ligand interaction can be restricted to tissue boundaries. Situations where ephrins and Ephs are co-expressed were expected to be dauntingly complex, considering the many possible cross-regulations proposed to occur and the potentially distinct pathways that each member of these families may stimulate. However, we found that the essence of the early boundaries can be represented by a surprisingly simple model where the final output can be largely predicted based on the relative selectivity of the extracellular interactions and the abundance of the various components.It is important to emphasize that the system cannot be simply reduced to the sum of binary inputs of individual specific pairs expressed in opposite tissues, but must rather be viewed as an integrated network made of semiselective pairs. Most of the molecules have more than one partner , and expression patterns range from equal distribution on both sides of the boundary to strong enrichment in one tissue, with intermediate partial asymmetries being most frequent. These characteristics of the network explain well its reaction to experimental manipulations: Although each component was required to a degree that generally corresponded to its expression levels and distribution, the specificity of the requirement was not absolute, as it could in some cases be substituted by another subtype that shared the same partner  can establish interactions of functional significance . This suAltogether, there data represent to our knowledge the most extensive dissection of network made of multiple ligands and receptors. Its success relied on the extensive use of combinations of loss- and gain-of function experiments comparing in parallel different ephrins and Ephs. Concerns often arise when attempting to evaluate the ability of one member of a gene family to substitute for another, due to the difficulty to control for the activity of the reagents. In this context, the strength of our experimental system resides in the high coherence of the systematic comparison of multiple conditions, including cross-rescues, where each reagent was validated in at least one of the complementary conditions e.g., .The comparison of three morphologically similar, cleft-like boundaries that form during Xenopus gastrulation and depend on Eph–ephrin signaling allowed us to extract recurrent regulatory motives in the form of complementary receptor–ligand pairs: ephrinB3—EphA4, ephrinB1–EphB2, and ephrinB2–EphB4 . For eacAltogether, the logic of ephrin/Eph-dependent repulsion regulation at tissue interfaces can be reconstructed as follows. For the complete cleft-like separation, a minimum of two antiparallel receptor–ligand pairs is required. If both receptors and both ligands were completely specific, signaling would be completely restricted to the boundary, as the ligands and receptors co-expressed in each tissue would interact only with partners in the adjacent tissue. If interactions were completely promiscuous, signaling intensity within each tissue were similar to that across the boundary, leading to the disintegration of the whole array. If interactions were relatively specific, favoring signaling between complementarily expressed pairs, repulsion could be restricted to the boundary by a threshold mechanism where full cell–cell detachment occurs only above a certain level of signaling intensity. In this situation, additional expression of receptors and ligands, with additional strong or weak interactions, would be compatible with proper boundary formation as long as signaling remained below the threshold within each tissue, and exceeded it at the tissue interface. This third scenario is the one that we encountered in the embryo. Such a dynamic yet robust system appears perfectly suited for the complex morphogenesis of vertebrate embryos. For example, they allow combining multiple functions of ephrin/Eph signaling, in the same tissues  and the University of Toronto Animal Care Committee .Affinity-purified antibodies specific for EphB4 and EphB2 were generous gifts from Dr. Elena Pasquale  comprising the extracellular domain of ephrin/Eph fused to C-terminal 6× histidin-tagged Fc region of human IgG were preclustered by 1 h incubation with anti-human Fc antibody (Jackson ImmunoResearch Laboratories) at a 1∶2 ratio in MBSH Membrane-targeted GFP and Cherry and Xenopus ephrinB1 and ephrinB2 were described previously To construct Eph chimeras with swapped cytoplasmic domains, a restriction site was introduced at the end of the transmembrane domain in each original receptor (Nhe1 for EphB4 and Spe1 for EphA4) by site-directed mutagenesis . The two resulting mutants, called EphA4* and EphB4*, had, respectively, a change from bp1926 tgtcat to actagt and from bp 1763 ggtggt to gctagc corresponding to a V to L and V to A substitution corresponding to the last hydrophobic amino acid of the transmembrane domain amino acid 558–559 of Xenopus EphB4 and EphA4. EphA4* was subcloned into pCS2+ for consistency with EphB4. EphA4* and EphB4* in pCS2+ rescued loss of the corresponding endogenous Ephs with the same efficiency as the original A4 and B4 constructs . The newmRNAs were synthesized according to the manufacturer's instructions . MOs and mRNAs were injected animally in the two blastomeres of two-cell stage embryos to target the ectoderm, and equatorially in the two dorsal blastomeres of four-cell stage embryos to target the mesoderm. MO sequences. The amounts of MO and of mRNA injected are listed in the mRNA was injected animally at the two-cell stage for ectoderm expression and dorsally at the four-cell stage for dorsal mesoderm expression. Dissections and assays were performed in Modified Barth Solution (MBSH) containing: 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 10 mM Hepes, adjusted to pH 7.4 with NaOH and supplemented with 10 µg/ml streptomycin sulfate and penicillin. For the standard assay, explants were dissected at stage 10+ (early gastrula). Ectoderm or mesoderm aggregates were laid on ectoderm caps, and the degree of separation was scored as the percentage of aggregates that did not incorporate into the cap after 45–60 min incubation t test.Separation of ventral tissue was similarly performed using stage 11 ectoderm and mesoderm from the ventral lip. For in vitro activation using soluble ephrins/Ephs, explants were either preincubated with preclustered ephrinB/Eph-Fc fragments (40 nM unless specified otherwise) in MBSH for 15 min at room temperature e.g.,  or the eCells from dissected mesoderm and inner layer ectoderm were dissociated in alkaline calcium-free buffer . For reaggregation, cells were transferred to agarose-coated Petri dishes containing MBSH and incubated for 1 h under mild rotation (10 rpm) on an orbital shaker. Images were taken under a dissecting microscope at a12× magnification using a Micropublished RTV3.3 camera (Qimaging) and were analyzed for object size using ImageJ software. Two parameters were measured: average object area and area/perimeter ratio. Results of six independent experiments were normalized using wild-type ectoderm or mesoderm as reference.Wild-type embryos were fixed at stage 10.5, and sagittal cryosections were prepared as described Tissues expressing membrane-targeted Cherry or GFP as well as various mRNAs/MOs were dissociated in dissociation buffer, cells were transferred to glass-bottom petri dishes  coated with 0.01 mg/ml Fibronectin, and cell behavior was filmed for 2 h. Cells were imaged with a WaveFX spinning disc confocal (Quorum Technologies) mounted on an automated DMI6000B Leica microscope, controlled with Volocity 3DM software (Improvision), using a 40× HCX PL APO CS, NA = 1.25 oil objective. Images were acquired every 2 to 5 min using an EM CCD 512×512 BT camera. Image processing was performed with Metamorph  and Adobe Photoshop7 software. Processing consisted of merging two to three planes from z stacks, assigning pseudo-colors, and adjusting image contrast.Cells from 14 ectoderm- and mesoderm-dissected explants were dissociated and an equal amount of cells were reaggregated either as mixed ectoderm/mesoderm aggregates, or as separate ectoderm and mesoderm aggregates for 1 h on agarose-coated plates in 1× MBSH. Mixed aggregates formed both homotypic ectoderm–ectoderm and mesoderm–mesoderm contacts as well as heterotypic ectoderm–mesoderm contacts, which mimicked the contacts at the boundary. The reaggregation time was set to maximize heterotypic contacts. Separate ectoderm and mesoderm aggregates, which formed only homotypic contacts, were combined for extraction, thus yielding the same amount of material as the mixed aggregates, and served as control. Extraction was performed in 1% NP40 containing buffer as previously described Ectoderm tissues were dissected from wild-type 40 embryos (for EphB2 and EphB4 immunoprecipiation) or from 40 embryos injected with EphA4-YFP mRNA (250 pg per blastomere) for EphA4 immunoprecipitation. Dissected tissues were treated for 40 min with an equal concentration (40 nM) of EphrinB1/B2/B3-Fc fragments, or control goat anti-human Fc. Tissues were extracted in 10 mM Hepes, 150 mM Nacl, 2 mM EDTA, 1% NP40, supplemented with protease and phosphatase inhibitors 2 buffer (Invitrogen), and 5 µl of a 6µM solution of primers. Cycling conditions were as follows: 94°C for 15 s, and 58°C for 30 s, 72.0°C for 1 min. Quantification was based on a dilution series (five fold steps) of the whole embryo RT. Relative expression levels were normalized as ratio to ODC, a ubiquitous gene with homogenous distribution in Xenopus embryos. The sequences of the primers are listed in RT-PCR was performed using mRNA extracted from whole-stage 9–12 embryos. Loading was equalized by comparing levels of FGFR. Two independent experiments showed identical temporal patterns of expression. RT-qPCR was performed using mRNA extracted from ectoderm and dorsal mesoderm at stage 10.5. qPCR was carried out using CFX 96 Thermo cycler (Biorad). The PCR reactions were set up using 5 µl of RT (20 to 50 times diluted) with 5 µl of SYBR green (Biorad) ½ dilution, 5 µl of 3× PCR-MgClFigure S1EphrinB1-3, EphA4, and EphB1–4 temporal expression during early Xenopus development and their relative tissue distribution. (A) General profile of total ephrin/Eph expression. RT-PCR was performed using mRNA extracted from whole embryos of the indicated stages. EphrinB1 and EphB1–4 are maternally expressed. EphrinB2, ephrinB3, and EphA4 are exclusively zygotic, starting at the onset of gastrulation (arrow). (B) Real-time quantitative RT-PCR of dissected tissues from stage 10.5 dorsal ectoderm and mesoderm, stage 11.5 ventral ectoderm and mesoderm, and stage 14 notochord and presomitic mesoderm. Bars express distribution between the two tissues. Error bars correspond to standard deviations (two independent series of samples). Numbers below each graph correspond to relative mRNA levels (arbitrary units), directly comparing all ephrins and Eph receptors for various tissues and stages. All values were corrected based on PCR efficiency. Average efficiencies are given above as %, with standard deviation.(TIF)Click here for additional data file.Figure S2Multiple Ephrin/Eph play an additive role in tissue separation across the boundary. (A) Individual and multiple knockdowns. Single MO injections for each ephrin or Eph yielded a mixing phenotype, the penetrance of which related to the relative enrichment in each tissue (compare to p<0.05 and p<0.01 (Student's t test) compared to controls (grey columns). (B) Each ephrin/Eph is specifically required and not replaceable. Individual ephrins and Eph receptors were depleted in the ectoderm or in the mesoderm, which induced inhibition of separation (white columns). Separation could be fully rescued by coinjection of mRNA (amounts indicated as pg/injection) coding for the corresponding ephrin/Eph (same colors). Only partial rescue was observed after heterotypical expression of other forms, even when expressed at high levels. * and ** indicate, respectively, p<0.05 and p<0.01 (Student's t test) compared to corresponding controls (white columns). “ns,” not significant. (C and D) Comparison by Western blot of ephrin levels in wild-type and manipulated ectoderm. (C) Conditions corresponding to the experiment presented in (B). Arrow points at specific ephrin band, decreasing in eB1MO. Both bands increased in ephrinB1/3 mRNA-injected embryos. Tubulin was used as the loading control. This blot is representative of three independent experiments. (D) Single and multiple ephrin depletion. Conditions are as in (TIF)Click here for additional data file.Figure S3Expression of EphA4/B4 chimera constructs. (A) Immunofluorescence. Sections from ectoderm tissues expressing the AB or BA chimeras (see (TIF)Click here for additional data file.Figure S4Eph kinase activity is required for tissue separation. (A) KD variants of EphA4 (EphA4KD) and EphB4 (EphB4 KD) act as dominant negatives. EphA4KD expression in the mesoderm inhibited tissue separation and failed to rescue EphA4 depletion. Identical results were obtained by expression of EphB4KD in the ectoderm. (B) Ectopic induction of tissue separation between ectoderm explants by ephrinB2 Fc treatment was blocked by expression of KD EphB4. (B′) Induction of separation between mesoderm explants by ephrinB3 Fc treatment was similarly inhibited by expression of EphA4KD. ** indicates p<0.01 (Student's t test) compared to second columns. (C) Inhibition of separation by ephrinB2 depletion in the mesoderm can be rescued by treatment of the ectoderm with soluble ephrinB2 fragments (see p<0.01 (Student's t test) compared to the first columns. “ns,” not significant. (D) Inhibition of Eph phosphorylation. Left panel, EphB4. Control and EphB4KD-expressing ectoderm explants were treated with soluble ephrinB2 Fc fragments. Extracts were prepared and analyzed by immunoblot for p-EphB, total EphB4, and tubulin. Stimulation of EphB phosphorylation by ephinB2 Fc fragments was strongly inhibited by expression of EphB4KD. Right panel, EphA4. Significant phosphorylation of EphA4 was observed in untreated mesoderm explants, consistent with activation by one of its endogenous ligands, ephrinB2 Click here for additional data file.Figure S5Effect of cadherin levels and ephrin-Eph signaling on separation and tissue cohesion. (A) Inhibition of separation upon cadherin overexpression and myosin inhibition. Tissue separation was inhibited by cadherin overexpression in the mesoderm but was rescued by increasing Eph signaling by treatment with soluble ephrinB2 Fc fragments. Separation was also strongly inhibited by treatment of wild-type explants with the myosin inhibitor blebbistatin. (B) Cadherin levels are not affected by Eph depletion. Immunofluorescence for C-cadherin of cryosections from whole embryos injected with control or anti-Eph morpholinos. GFP (immunostained in red) was used as the tracer. Note the strong disruption of the ectoderm–mesoderm boundary. (C–E) Tissue cohesion is decreased upon cadherin depletion or ectopic ephrin/Eph expression. Dissociated ectoderm and mesoderm cells were left to reaggregate under mild rotation for 1 h. (C) Effect of cadherin and/or EphB4 depletion on mesoderm reaggregation. (D) Effect of ephrin/Eph ectopic expression on mesoderm or ectoderm reaggregation. Ectoderm-specific ephrinB3 and EphB4 were expressed in the mesoderm, and mesoderm-specific ephrinB2 and EphA4 in the ectoderm. (E) Quantification of reaggregation. Two criteria were used, which gave similar results: the average aggregate area, which reflects the extent of aggregation, and area/perimeter ratio, which integrates both the size of the aggregates and their degree of compaction. Results from individual experiments were normalized using wild-type ectoderm/mesoderm as the reference (1.0) to account for batch-to-batch variation.(TIF)Click here for additional data file.Figure S6Dose response of Eph activation by soluble ephrins. Ectoderm explants were incubated for 30 min in the presence of different concentrations of ephrin-Fc fragments. Total extracts were analyzed by Western blot for p-Tyrosin levels. Phosphorylated Ephs represent a prominent band around 110 kDa (arrow) Click here for additional data file.Figure S7Simulation of ephrin/Eph signaling in dorsal ectoderm and mesoderm and at the boundary. (A) Principle of the simulation: the total signal output due to all the interactions between ephrins and Eph receptors at the tissue interface is computed (red double arrow), also taking into account the involvement of these molecules at homotypic contacts with surrounding cells in each tissue . (A′) Diagrams of all the high affinity interactions between ephrins and Eph receptors at different cell contacts. Relative concentrations are symbolized by the size of the boxes, whereas the thickness of the red lines represents the relative intensities of the individual signals. (B and C) Apparent affinities and concentrations used for the simulation . (D) Effect of varying the range of concentrations and affinities on output for the stage 10.5 dorsal boundary. Each range of concentration was obtained by multiplying the values of table C by the indicated value. Selected affinities were varied as indicated. All other values were as in table B. The condition marked by a star corresponds to the basal values of tables B and C. (E) Results from (TIF)Click here for additional data file.Figure S8Effect of ephrin/Eph gain- and loss-of-function on ventral ectoderm–mesoderm separation. (A) Summary of ephrin/Eph expression in stage 11 ventral tissues. The major differences compared to the dorsal side (p<0.01 (Student's t test) compared to corresponding controls (white columns). (C) Induction of separation. Control ventral ectoderm explants normally mix. Significant separation was observed upon explant treatment with soluble Fc fragments corresponding to mesoderm-enriched ephrinB1 and B2, but not ephrinB3. EphrinB1-Fc–induced separation was significantly inhibited by EphB2 depletion, but not EphB4 depletion. The result is consistent with EphB2 acting as the preferred receptor for ephrinB1 Click here for additional data file.Movie S1Live imaging of contacts between single ectoderm and mesoderm cells. A mixture of dissociated wild-type ectoderm cells expressing membrane-targeted GFP (green) and mesoderm cells expressing membrane-targeted Cherry (red) was plated on fibronectin. Newly re-established homotypic contacts between cells of the same tissue remained stable. Contacts between ectoderm and mesoderm cells underwent cycles of attachment (arrowheads) and repulsion/detachment (arrows). This movie shows maximum projection of 3 z planes of 0.2 µm distance.(MP4)Click here for additional data file.Movie S2Live imaging of contact between single ectoderm and mesoderm cells treated with blebbistatin. Mesoderm cells (red) and ectoderm cells (green) treated with 100 µM blebbistatin formed stable heterotypic contacts.(MP4)Click here for additional data file.Movie S3Live imaging of contacts between single ectoderm and mesoderm cells overexpressing cadherin. Mesoderm cells overexpressing cadherin (red) and ectoderm cells (green) maintained stable heterotypic contacts.(MP4)Click here for additional data file.Movie S4Live imaging of contacts between control mesoderm cells. Examples of formation of stable contacts between wild-type mesoderm cells (arrowheads).(MP4)Click here for additional data file.Movie S5Live imaging of contacts between cadherin-depleted mesoderm cells. Mesoderm cells injected with cadherin MO failed to establish stable contacts. The cell on the bottom right attaches (arrowheads) and detaches (arrows) several times from the neighboring cells. Arrowheads point to attachments, arrows to detachments.(MP4)Click here for additional data file.Movie S6Live imaging of contacts between cadherin/EphB4-depleted mesoderm cells. Mesoderm cells were prepared from embryos coinjected with cadherin MO and EphB4MO. Cells formed contact (arrowheads) and failed to retract. This demonstrates that the repulsion observed between cadherin MO cells was due to ephrin–Eph signaling.(MP4)Click here for additional data file.Movie S7Live imaging of contacts between mesoderm cells overexpressing ephrinB3 and EphB4. Ectopic coexpression of “ectodermal” ephrinB3 and EphB4 induced cycles of attachment and detachments between mesoderm cells. Arrowheads mark sites of contact and arrows subsequent retractions between the cell on the right side of the field and the two neighboring cells in the middle.(MP4)Click here for additional data file.Text S1Supplementary materials and methods, and description of the simulation model of ephrin/Eph signaling.(DOCX)Click here for additional data file.


Postnatal neural progenitor cells of the enteric nervous system are a potential source for future cell replacement therapies of developmental dysplasia like Hirschsprung's disease. However, little is known about the molecular mechanisms driving the homeostasis and differentiation of this cell pool. In this work, we conducted Affymetrix GeneChip experiments to identify differences in gene regulation between proliferation and early differentiation of enteric neural progenitors from neonatal mice. We detected a total of 1333 regulated genes that were linked to different groups of cellular mechanisms involved in cell cycle, apoptosis, neural proliferation, and differentiation. As expected, we found an augmented inhibition in the gene expression of cell cycle progression as well as an enhanced mRNA expression of neuronal and glial differentiation markers. We further found a marked inactivation of the canonical Wnt pathway after the induction of cellular differentiation. Taken together, these data demonstrate the various molecular mechanisms taking place during the proliferation and early differentiation of enteric neural progenitor cells. The enteric nervous system (ENS) is a largely autonomous and highly complex neuronal network found in the gastrointestinal tract. Its two major plexuses are integrated into the layered anatomy of the gut wall and, together with central modulating influences, exert control over gastrointestinal motility, secretion, ion-homeostasis, and immunological mechanisms . In orde in vitro expansion of enteric neural stem cells derived from small biopsy materials. However, we are just beginning to understand the molecular mechanisms that underlie neural stem cell biology and how this knowledge can be used for optimizing in vitro culture conditions [HSCR is hallmarked by an aganglionic distal bowel leading to life-threatening disturbances in intestinal motility. Today's therapeutic gold standard, the surgical resection of the affected gut segments, is nevertheless associated with problematic long-term outcomes with regard to continence . In ordenditions , 12. in vitro [Genome-wide gene-expression analyses are a useful tool to examine the genetic programs and cellular interactions and have been widely used to identify potential markers or signalling mechanisms especially in CNS neurospheres or cancer tissues. Further, gene-expression assays have also helped to unravel genetic prepositions associated with HSCR , 14, thoin vitro . in vitro.Here, we used an Affymetrix microarray analysis to evaluate the genetic expression profile of proliferating murine enteric neural stem cells and its changes during the early differentiationCell culturing was conducted as described previously . The hanμg/mL; Roche Diagnostics, Mannheim, Germany) dissolved in Hanks' balanced salt solution with Ca2+/Mg2+  for 30 min at 37°C. During enzymatic dissociation the tissue was carefully triturated every 10 min with a fire polished 1 mL pipette tip. Prior to the first trituration step, cell suspension was treated with 0.05% (w/v) DNAse I (Sigma-Aldrich). After 30 min, tissue dissociation was stopped by adding fetal calf serum  to a final concentration of 10% (v/v) to the medium. Undigested larger tissue pieces were removed with a 40 μm cell strainer . Residual enzymes were removed during two washing steps in HBSS at 200 g. After dissociation, cells were resuspended in proliferation culture medium ) containing N2 supplement , penicillin , streptomycin , L-glutamine , epidermal growth factor , and fibroblast growth factor . Cells were seeded into 6-well plates (BD Biosciences) in a concentration of 2.5 × 104 cells/cm2. Only once before seeding, the medium was supplemented with B27 . EGF and FGF were added daily and culture medium was exchanged every 3 days. All cultivation steps were conducted in a humidified incubator at 37°C and 5% CO2. An overview of the following cell culture protocol is shown in Neonatal (P0) C57BL/6 mice without regard to sex were decapitated and the whole gut was removed. After removal of adherent mesentery the longitudinal and circular muscle layers containing myenteric plexus could be stripped as a whole from the small intestine. Tissue was chopped and incubated in collagenase type XI  and dispase II (250 μg/mL), L-glutamine (2 mM), and ascorbic acid-2-phosphate .Single free-floating enterospheres (50 enterospheres/dish) were picked again, washed 3 times in Tris buffer, and transferred into new petri dishes containing either proliferation medium or differentiation medium. Differentiation medium consists of DMEM/F12 containing N2 supplement (1 : 100), penicillin (100 U/mL), streptomycin  was successively compared by microarray analysis as described below.Affymetrix microarray analysis was conducted similar to previously published data in three independent experiments, each with cell cultures prepared from 2 pups from the same litter . In eachTotal RNA of enterospheres of both groups was extracted using the RNeasy Micro Kit (Qiagen). RNA quality was evaluated on Agilent 2100 Bioanalyzer with RNA integrity numbers (RIN) of the samples in this study being in the range from 8 to 10. RIN numbers higher than 8 are considered optimal for downstream application .μg of labeled and fragmented cDNA was hybridized to GeneChip Mouse Gene 1.0 ST arrays (Affymetrix). After hybridization, the arrays were stained and washed in a Fluidics Station 450 (Affymetrix) with the recommended washing procedure. Biotinylated cDNA bound to target molecules was detected with streptavidin-coupled phycoerythrin, biotinylated anti-streptavidin IgG antibodies and again streptavidin-coupled phycoerythrin according to the protocol. Arrays were scanned using the GCS3000 GeneChip Scanner (Affymetrix) and AGCC 3.0 software. Scanned images were subjected to visual inspection to check for hybridization artifacts and proper grid alignment and analyzed with Expression Console 1.0 (Affymetrix) to generate report files for quality control.Double-stranded cDNA was synthesized from 100 ng of total RNA, subsequently linearly amplified, and biotinylated using the GeneChip WT cDNA Synthesis and Amplification Kit  according to the manufacturer's instructions. 15 t-test without multiple testing correction (Partek), selecting all transcripts with a minimum change in expression level of 1.5-fold together with a p value less than 0.05.Normalization of raw data was performed by the Partek Software 6.6, applying an RMA (Robust Multichip Average) algorithm. Significance was calculated using a  in vitro. Therefore, we generated enterospheres by 9 day in vitro cultures, which then could be picked and either proliferated or differentiated for two more days .In total, the gene chip detected 1454 transcripts to be at least 1.5-fold differentially expressed between proliferating and differentiating enterospheres. 1333 of these transcripts code for already identified proteins. 541 genes were found to be upregulated and 792 genes were found to be downregulated in comparison to proliferating enterospheres  and data mining with the science literature search engine poptosis . It is npoptosis  as well poptosis . Additiopoptosis , Table 5 in vitro cultures of enteric neural progenitor cells derived from murine tunica muscularis to scan for molecular programs and signalling pathways acting on cell proliferation and early differentiation.The proliferation and differentiation of enteric neural progenitor cells during embryonic and postnatal development are controlled by a complex interplay of various intrinsic and extrinsic factors. Their exact timing is crucial for proper migration and proliferation of neural crest cells and for their differentiation into the various neural cell types that compose the complex neural structures of the ENS. Although research in recent years extended our understanding of ENS development and its pathologies , there aOur experiment aimed to elucidate gene regulations in enterospheres that occur while ENS progenitor cells leave their proliferative state and begin to differentiate into more defined and specific cell types. The results of the Affymetrix gene expression analysis showed the up- and downregulation of overall 1333 known genes that code for already identified proteins. 171 of these genes could be linked to cell proliferation . Interes in vivo and require further investigation. Together, on a broad basis, this dataset provides strong evidence that this cell culture design is applicable to decreasing the proliferative rate of enteric neural progenitor cells without inducing cell death or apoptosis in an appreciable quantity.We also checked the regulated genes for apoptosis markers to see whether the stop in proliferation was related to cell death (Supplementary Table 2). Since only 3 of 12 apoptotic genes were regulated in the direction that indicates apoptosis, it is unlikely that apoptosis played a leading role in the interruption of proliferation. Still, the effect and regulation of apoptosis during enteric sphere cultures are an important cornerstone of understanding enteric neural progenitors in culture and in vivo. Interestingly, we found 8 genes involved in adult central or embryonic neural stem cells homeostasis . Therefore, β-catenin accumulates in the cytoplasm and translocates to the nucleus where it binds to TCF/LEF transcription factors to initiate Wnt target gene expression. Interestingly, our current data strongly indicate that the canonical Wnt pathway is switched off during the first two days of enteric progenitor differentiation on several levels of the signalling cascade , in thend LRRK2 , NKD1 [5nd LRRK2 , PRICKLEnd LRRK2 , NOV [56nd LRRK2 ), or in nd LRRK2 , EDIL3 [nd LRRK2 ). Furthend LRRK2 . We alsond LRRK2 , a Wnt tnd LRRK2 , in the nd LRRK2 . in vitro analyses strongly support this hypothesis.Taken together, it is conceivable that canonical Wnt signalling plays a role in the maintenance of the enteric progenitor pool during proliferation and is switched off at the beginning of differentiation conditions. Indeed, our previous gene expression analyses  as well  in vitro. Using microarray analysis, we found a marked inhibition of cell cycle progression in general as well as strong evidence for neural stem cells differentiation into enteric neurons and glia cells. These findings were substantiated by the upregulation of genes related to synapse formation and neural connectivity. Most interesting, we found that this transition from enteric neural progenitor proliferation to differentiation was accompanied by a considerable inactivation of the canonical Wnt signalling pathway. This, together with previous work, strongly indicates that canonical Wnt activation is one of the driving mechanisms of enteric neural progenitor proliferation and thus might play a role in the homeostasis of this cell pool in vivo and in vitro.This study focused on the changes in gene expression of enteric neural progenitor cells occurring within the first two days of transition from a proliferative state to differentiationThe supplementary material contains tables with additional information about the gene expression analysis.


The recovered genomes belonged to Leifsonia (n = 2), Rhodanobacter (n = 5), Acidobacteria (n = 2), Sporolactobacillus , Ktedonobacter , Streptomyces , and Burkholderia . Assembled genomes averaged to 5.9 Mb, with relative abundances ranging from rare (<0.0001%) to relatively abundant (>0.01%) in the original soil microbiome. Furthermore, we detected them in samples collected from geographically distant locations, particularly more in temperate soils compared to samples originating from high-latitude soils and deserts. To the best of our knowledge, this study is the first successful attempt to assemble multiple bacterial genomes directly from a soil sample. Our findings demonstrate that developing pertinent enrichment conditions can stimulate environmental genomic discoveries that would have been impossible to achieve with canonical approaches that focus solely upon post-sequencing data treatment.Despite extensive direct sequencing efforts and advanced analytical tools, reconstructing microbial genomes from soil using metagenomics have been challenging due to the tremendous diversity and relatively uniform distribution of genomes found in this system. Here we used enrichment techniques in an attempt to decrease the complexity of a soil microbiome prior to sequencing by submitting it to a range of physical and chemical stresses in 23 separate microcosms for 4 months. The metagenomic analysis of these microcosms at the end of the treatment yielded 540 Mb of assembly using standard  Soil microbial communities might display the highest level of bacterial diversity of any environment with a single gram reported to contain about a billion cells making up thousands to millions of different taxa  have been developed to by-pass the limitations associated with cultivation efforts  and bioprospecting  was sprayed for mercury enrichment #1; ESC 10: 5 ml of purified water with inorganic mercury  was sprayed for mercury enrichment #2. Note that microcosms were aerated (by opening the cap for about a minute) every week to renewing the normal air condition. For the nitrogen condition (ESC 5), this step was immediately followed by replacing the air with nitrogen gas.Microcosms were done in triplicates (50 g of soil in each microcosm), stored at room temperature (except for the high temperature condition), without light, and were hermetically closed during the experiment. Following ESCs applied to each microcosm: ESC C (control): 5 ml of purified water was sprayed for control; ESC 1: 5 ml of purified water with ethanol (20% of the volume) was sprayed for ethanol enrichment; ESC 2: 5 ml of purified water with NaCl (30 g/L) was sprayed for salt enrichment #1; ESC 3: 5 ml of purified water with NaCl  was sprayed for salt enrichment #2; ESC 4: 5 ml of purified water was sprayed and then microcosms were incubated at 37°C for high temperature condition; ESC 5: 5 ml of purified water was sprayed and then, the microcosm atmosphere was replaced by nitrogen gas for nitrogen atmospheric condition; ESC 6: 5 ml of purified water with diesel  was sprayed for diesel enrichment; ESC 7: 5 ml of purified water with heavy metals  was sprayed for heavy metals enrichment #1; ESC 8: 5 ml of purified water with heavy metals  was sprayed for heavy metals enrichment #2; ESC 9: 5 ml of purified water with inorganic mercury , a protocol known to be relatively efficient for this particular soil . In a first phase, pair-end sequencing was done on duplicate microcosms of each ESC (about one million reads per microcosm). As an exception, the three replicates of the mercury enrichment #2 were sequenced due to a low reproducibility of one microcosm observed using RISA profiles. Thus, 23 metagenomic data were generated. In a second phase, a mate-pair sequencing effort with 3 kb of gap was done for duplicates corresponding to 3 promising ESCs: heavy metals enrichment #2 (one million reads each), mercury enrichment #1 (two million reads each) and the outlier replicate of the mercury enrichment #2 (one million reads). Duplicates corresponding to the ethanol enrichment were also further sequenced, however, due to the high fragmentation of the DNA recovered from this ESC, additional sequencing was done with paired-end library preparation instead of mate-pair.Streptomyces, 18 Chloroflexi, 10 Acidobacteria, 6 Rhodanobacter, and 3 Sporolactobacillus. In parallel, sequences related to the 16S rRNA gene were screened from metagenomic datasets using RDP  and pH (5′ AAG GAG GTG ATC CAG CCG CA 3′) were used for this amplification. PCR products were then gel-purified  and cloned in a TOPO-TA using the TOPO-TA cloning kit for Sequencing (Invitrogen). 96 clones for each microcosm condition were randomly selected and inoculated in a 96 well plate. After TOPO-TA vector extraction, inserts were sequenced with Sanger Technology (Beckmann Coulter Genomics). Both primer M13for (5′ TGT AAA ACG ACG GCC AGT 3) and primer M13rev (5′ CAG GAA ACA GCT ATG ACC 3′) reactions were done. Sequences obtained with forward and reverse reactions were assembled and vector-purified with Seqman Software (Lasergen Seqman DNASTAR). Consensus sequences were then compiled by dataset for further data analysis.Genomic taxonomical affiliation was inferred using information extracted from (i) global sequence similarity with the RAST collection of genomes , indicating that the microcosm strategy initiated a shift in the composition of the initial microbial community . We detected positive selective pressure effect on functions directly related to ESCs: the Pfam related to Cobalt-zinc-cadmium resistance protein CzcD was more prevalent under the heavy metal enrichment #2, anaerobic cytochrome c552 was higher in soil under nitrogen gas atmosphere, and mercury resistance operon regulatory protein was higher in the mercury enrichments. Moreover, some functions were reproducibly detected only under specific conditions, such as IncI1 plasmid conjugative transfer DNA primase in microcosms from the heavy metal enrichment #2.We sequenced the genetic material of two microcosms for each ESC as well as the outlier microcosm replicate of the mercury enrichment #2  based on metagenomic assembly scores Figure . In mostLeifsonia (n = 2), Rhodanobacter (n = 5), Acidobacteria (n = 2), Sporolactobacillus (n = 2), Ktedonobacter (n = 1), Streptomyces (n = 3), and Burkholderia (n = 2). They carry between 2,637 and 9,051 genes and possess a global GC-content ranging from 47.3% to 71%. Our analysis of single-copy gene families revealed that 11 of 17 bacterial genomes with no close relatives in their microcosms were 97% complete in average (Table Streptomyces genomes and the two Burkholderia genomes) estimated that they were 61% complete in average, despite their large sizes . As an alternative approach we compared these draft genomes to the best matching reference genomes available using the best-hit function implemented in RAST. Our analysis indicated that the two Streptomyces draft genomes (Mer-2-A and Mer-2-B) covered 81% and 83% of the gold standard genome Streptomyces avermitilis MA-4680, respectively. Similarly, although single-copy gene analysis estimated only 45% and 36% completion for the two Burkholderia draft genomes, they covered 87–90% of the gold standard Burkholderia xenovorans LB400 chromosome 1. These contrasting findings suggest that draft genome completion estimates based on single-copy gene families can underestimate the rate of recovery when multiple closely related bacterial genomes are present in the assembly.We determined a total of 17 draft genomes from these microcosms. Table Ktedonobacteraceae. Until now, this lineage had only one representative genome sequenced due to the difficulty cultivating this branch of the tree of life  using G+C skew and recovered two replicons and a megaplasmid for each of them  and no differences could be detected between their full-length 16S rRNA genes. On the other hand, most genes and functions were not shared between the two mega-plasmids  further supports this suggestion. Similar operons have been found in plasmids worldwide  in lower abundance. Overall, each ESC provided a unique set of genomes, suggesting that more genomes could be recovered by applying additional conditions to this soil. The recovery of distinct organisms by only changing the concentration of mercury also suggests that a fine gradient of concentrations may stimulate the recovery of more genomes from organisms that prefer a specific range. It is indeed difficult to estimate what fraction of the soil microbial community that can be targeted from such enrichment approaches, yet ESCs may be useful to build a larger catalog of genes and their genomic contexts found in soil.Different taxonomic groups in our soil sample adapted to different ESCs. For instance, while ethanol stimulated the growth of Firmicutes, heavy metals stimulated Proteobacteria and Actinobacteria. We generated a tetranucleotide frequency-based tree to display the distribution of 8 taxonomical groups that were present in our microcosms Figure . RecoverSporolactobacillus), as well as smaller, microcosm-specific ones . Micro-diversity traits are commonly found in cultivated strains of similar taxonomy, including in those displaying identical 16S rRNA genes . Each taxonomical group possessed unique functions .We performed a network visualization using observed functions in recovered genomes from each ESC Figure . SimilarSporolactobacillus genomes possessed more than 40 genes related to spore formation, none of these genes being detected in the other genomes. Derived from the same condition and representing an unusual functional trait for Bacteria, the Ktedonobacter genome has the potential to produce both vitamin K1 (Phylloquinone) and K2 (Menaquinone) from Chorismate. The three Streptomyces genomes carried genes related to ethylmalonyl-CoA pathway of C2 assimilation, spore pigment biosynthetic cluster, sigmaB stress response regulation, sialic acid metabolism and siderophore assembly. As a potential industrial interest, several novel polyketide synthase modules were detected in these genomes. The two Burkholderia species possessed genes for degrading benzoate, but only one was carrying genes for a chloroaromatic degradation pathway, while the other harbored genes for degrading chlorobenzoate. When focusing on the nitrogen cycle, all these populations had the functional potential to assimilate ammonia and most of them could perform nitrite and nitrate ammonification. On the other hand, only the Sporolactobacillus genomes possessed adequate genetic information to fix nitrogen from the atmosphere (operon nifDKHENBQU). This is the first time this taxon is linked to nitrogen fixation, which is an important ecological trait in soil systems  with distinct functions. A majority of these organisms possessed gliding and flagellar motility capacities. Moreover, they harbored different metabolic capacities  and strategies to target iron  Table . The SpoRhodanobacter genomes recovered from our ESCs contained more than a thousand additional functions compared to the six culture-derived Rhodanobacter genomes currently available. These functions include secretion pathway proteins, membrane and outer membrane proteins, peptidases, a penicillin amidase, various transcriptional regulators, virulence proteins, chitin utilization and lignin degradation proteins as well as proteins related to cyanophycin metabolism (both cyanophycin synthase and cyanophycinase) and inositol catabolism. On the other hand, 997 functions were detected in all Rhodanobacter genomes and 1159 only in the culture-derived genomes. The network analysis displayed separate groupings of cultivated and metagenomic analysis-based Rhodanobacter genomes, possibly due to differences in methodologies , providing clear geographic patterns for the recruited soil microbiome fraction. We subsequently used these genetic structures  as a first soil reference database of its kind to classify soil biomes and defined tree groups depicting deserts, high latitude soils and temperate soils  from a soil sample using enrichment strategies. The assembled genetic material constituted about 1% of the original soil microbiome, and we achieved this using about 10 Gb of sequencing and standard bioinformatics approaches. We detected these draft genomes also in soil samples collected from distant locations. Although the recovered genetic structures from Park Grass explain only 1% of the diversity, considering the absence of any genomes recovered from soil through metagenomics to date, our study demonstrates the efficacy of pre-sequencing enrichment applications as a way to break into the vastly unexplored soil microbiome.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Therapeutic drugs for cognitive and psychiatric disorders are often characterized by their molecular mechanism of action. Here we demonstrate a new approach to elucidate drug action on large-scale neuronal activity by tracking somatic calcium dynamics in hundreds of CA1 hippocampal neurons of pharmacologically manipulated behaving mice. We used an adeno-associated viral vector to express the calcium sensor GCaMP3 in CA1 pyramidal cells under control of the CaMKII promoter and a miniaturized microscope to observe cellular dynamics. We visualized these dynamics with and without a systemic administration of Zolpidem, a GABAA agonist that is the most commonly prescribed drug for the treatment of insomnia in the United States. Despite growing concerns about the potential adverse effects of Zolpidem on memory and cognition, it remained unclear whether Zolpidem alters neuronal activity in the hippocampus, a brain area critical for cognition and memory. Zolpidem, when delivered at a dose known to induce and prolong sleep, strongly suppressed CA1 calcium signaling. The rate of calcium transients after Zolpidem administration was significantly lower compared to vehicle treatment. To factor out the contribution of changes in locomotor or physiological conditions following Zolpidem treatment, we compared the cellular activity across comparable epochs matched by locomotor and physiological assessments. This analysis revealed significantly depressive effects of Zolpidem regardless of the animal’s state. Individual hippocampal CA1 pyramidal cells differed in their responses to Zolpidem with the majority (∼65%) significantly decreasing the rate of calcium transients, and a small subset (3%) showing an unexpected and significant increase. By linking molecular mechanisms with the dynamics of neural circuitry and behavioral states, this approach has the potential to contribute substantially to the development of new therapeutics for the treatment of CNS disorders. Understanding how drugs affect complex neuronal networks is critical for the future of neuroscience drug discovery. Therefore, it is essential to invest in the development and applications of new technologies that will enable researchers to study functional neuronal networks. Recently, the BRAIN initiative outlined a set of experimental techniques that hold the most promise to advance our understanding of brain function and brain disorders Imaging neuronal calcium dynamics in behaving animals with miniaturized integrated fluorescent microscopes takes advantage of several recent break-throughs in technology: using viral vectors to express fluorescent indicators in a targeted genetically identified neuronal population This technology, especially if used in combination with other recording techniques, is a transformative new platform for neuroscience drug discovery research. This powerful combination has the ability to efficiently identify compounds that either disrupt normal neuronal activity, or restore normal network activity that was affected by disease, stress or pharmacological manipulations. The traditional drug discovery process is based on a drug’s ability to affect isolated biological targets in artificial systems with subsequent validation in functional and behavioral assays. The latter is often uninformative because the same underlying changes in neuronal networks can have distinct species-specific behavioral effects; conversely, apparently similar behaviors can have different underlying causes. Despite the need to directly investigate drug effects on neuronal activity in freely behaving animals, this step was often omitted because, until recently, techniques lacked the necessary neuronal yield and were not easily integrated into industrial settings. High throughput in-vivo calcium imaging overcomes these limitations.To demonstrate the potential of this approach for drug discovery, we conducted a proof-of-concept study investigating the effects of Zolpidem on hippocampal neuronal activity measured with the genetically-encoded calcium indicator GCaMP3. Zolpidem was selected for these studies as it (a) is widely used as a therapeutic agent; (b) is pharmacologically well-characterized; (c) has a straightforward behavioral and physiological readout (sleep); (d) has not been characterized in terms of impact on the neuronal activity in behaving animals.in vivo, let alone in freely behaving animals.Zolpidem is a short-acting nonbenzodiazepine hypnotic that potentiates GABA transmission by acting on GABA A receptors  to track somatic calcium dynamics of hundreds of hippocampal neurons expressing the calcium indicator GCaMP3 under CamKII promoter. We selected a dose of Zolpidem (10 mg/kg) that closely reproduces sleep-inducing effects in humans: decreased wake time and latency to sleep with specific increases in non-rapid eye movement sleep (NREM) (reviewed in The University of Pennsylvania Penn Vector Core custom produced AAV2/5 vector expressing GCaMP3 (HHMI/Janelia Farm) via the CaMKII promoter . After imaging experiments, we verified the levels of GCaMP3 expression in CA1 pyramidal neurons  was dissolved in ultrapure water to 1 mg/ml. The mice were weighed before each imaging session and given an oral gavage of the same volume of vehicle (ultrapure water) and Zolpidem (10 mg/kg).3) directly above CA1was removed, without disturbing the structure being imaged. During the second surgical procedure, a subset of animals also received a subcutaneously implanted telemetric device  for polysomnographic recordings All animal experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals adopted by the US National Institutes of Health (Janssen IACUC protocol 100–311); the IACUC committee specifically approved this study. Male C56BL/6 mice, aged 8–12 weeks at start, were housed individually with enrichment under controlled conditions with lights on at 6 am, 12∶12 light/dark schedule. The mice underwent two separate surgical procedures under Isoflurane (1.5–2.5%) with analgesic treatment . During the first surgery, the viral vector was injected (900 nL) into CA1  . Upon full recovery (1–2 weeks later), an optical guide tube was implanted over CA1 The mice were allowed to fully recover (4–6 weeks) before the final preparation for the imaging experiments. First, under Isoflurane (1–1.5%) sedation, the lens  was affixed into the optical guide tube Prior to the actual imaging experiment, the mice were habituated for 3–6 days. The habituation sessions were identical to the actual imaging sessions except that the vehicle was dosed and a “dummy” scope  that mimics the actual microscope’s shape and weight was used.The timing of the imaging session is shown on The multimodal recording sessions were the same as in the open field with the following modifications: (1) the imaging was performed while animals were freely behaving in the animal’s home cage placed in a sleep recording chamber To determine states of vigilance, body temperature, EEG and EMG signals were recorded for 3 hour following vehicle and drug administration and the signals were then digitized (100 Hz sampling rate) using Dataquest A.R.T. software . EEG/EMG recordings were binned into 10 sec intervals and classified according to the vigilance state as previously described For the analysis of neuronal activity during NREM, the epochs were selected according to the following criteria: a 4 min bin of uninterrupted unambiguous NREM which consisted of 24 uninterrupted 10-sec bins classified as “NREM” and was flanked by at least 4 of consecutive 10-sec NREM bins. The alignment between the imaging system and multimodal data collection was done by recording, on a separate channel, the state of the imaging system transmitted through an analog channel in nVista and subsequently digitized at 100 Hz.An overhead camera connected to an automated video tracking system  on a PC computer recorded and digitized (30 Hz sampling rate) the x- and y-position in a 50 × 50 cm arena. The system automatically detected the large ambulatory movements (>7.2 cm/min), small movements (<7.2 cm and >0.2 cm/min) and inactivity (<0.2 cm/min). The velocity  was calculated in 1-sec bins. Alignment between the imaging and the video tracking systems was done by triggering imaging through the VideoTrack software.F′(t)/F′0 = (F′(t) – F′0)/F′0, where F′0 is the projected mean intensity for all frames. Spatial filters corresponding to individual cells were identified using a cell-sorting algorithm that applies principal and independent component analyses 2+ activity over the entire session. Each cell’s thresholded spatial filter was used to extract the individual cell’s Ca2+ activity from the ΔF′(t)/F′0 stack. The Ca2+ transients were identified by searching each trace for local maxima that had peak amplitude more than two s.d. from the baseline  for at least 0.5 sec at a separation of 300 ms from adjacent Ca2+ transients.A custom in-house Matlab script was used to preform image analysis as we briefly describe here. Since the original high-definition data provided finer spatial resolution than necessary for analysis of cellular activity, a spatial down-sampling of 16x was applied and the image cropped to visualize the area containing optimal cellular signal activity. Correction for the slight lateral displacement (motion artifact) was achieved by applying an image registration (ImageJ plugin TurboReg implemented under the MatLab interface) to align all movie frames to a single movie target. To identify the individual cells, the registered images were expressed as relative change in fluorescence, ΔThe calcium transients event rate for each individual cell was calculated across both treatment conditions (vehicle and Zolpidem) to determine the effect of Zolpidem on individual cells and population activity. To compare event rates across drug conditions (vehicle vs Zolpidem), the Wilcoxon Signed Rank Test (WSR) was used on the data pooled across cells and binned into 1-minute intervals. To take into account the number of animals used in the experiments, and the variations between animals, we also performed a one-tailed WSR tests on the averaged values obtained for each animal thus using sample “n” as number of animals rather than the number of cells. To compare neuronal activity during active and inactive periods, 1-minute intervals containing at least one large ambulatory movement (>7.2 cm/min displacements) were designated as active, while 1-minute intervals during which total displacements were less than 0.2 cm were designated as inactive. To study Zolpidem effects on the level of individual cells, the event rates  were compared using Mann-Whitney-Wilcoxon (MWW) test. Initially, cells with a significant change (p<0.05) were identified; these cells were then subdivided into two groups depending on the direction of change (increased or decreased event rate). To address the concern that the observed drug effects on the level of individual cells are not above the level expected by chance, we first calculated the normalized drug index for each cell: (post-drug event rate - post-vehicle event rate)/(post-drug event rate+post-vehicle event rate). The expected (if Zolpidem had no effect) distribution of drug indices was then constructed by re-sampling (with replacement) post-vehicle bins within each cell . A cell was considered to have a significant effect if its index value was outside the 99% confidence interval for the expected distribution.−4; non-significant effect of the protocol: “Telemetry only” vs. “Telemetry+Imaging”, p = 0.39; non-significant interaction, p = 0.34). In a comparable manner, the time spent in wake was reduced in “Telemetry only” (−38%) and “Telemetry+Imaging” (−44%) groups ; whereas NREM sleep duration was increased in “Telemetry only” (+29%) and “Telemetry+Imaging” (+46%) groups during the first 2 hours after dosing relative to vehicle treatment . Zolpidem also reduced locomotor activity in both groups . Changes in the REM sleep state were not significant following zolpidem dosing vs. vehicle in both groups, although a tendency toward an increase in REM latency and a decrease in REM duration were observed.To assess whether the imaging surgical preparations and imaging procedures  disrupted the sleep-promoting effects of Zolpidem, we conducted a study to compare the effects of Zolpidem in a group of animals in which we conducted telemetry (EEG/EMG and locomotor activity) recordings only (“Telemetry only”) to another group in which telemetry was conducted at the same time as calcium imaging (“Telemetry+Imaging”). When orally dosed at the beginning of the light phase, Zolpidem (10 mg/kg) was effective in inducing and prolonging sleep duration in both groups of mice. As shown in The timing of a typical experimental session used to collect imaging videos of the fluorescent cellular signal after oral administration of the vehicle (water) and Zolpidem (10 mg/kg) is shown on The animals’ locomotor activity was continuously monitored during the imaging sessions. Quantification of the animals’ displacements over the course of the session was carried out in 1 second time bins. In confirmation of previous studies, Zolpidem significantly lowered the animals’ locomotor activity . Correspondingly, there was a significant difference in frequencies of calcium transients between active and inactive periods in vehicle  and Zolpidem conditions  measured in 1-minute bins during which the animals were inactive; inactivity bins were defined as bins with total displacements of less than 0.2 cm. An example of a typical imaging session demonstrating this approach is shown on We assessed the changes in rate of calcium transients between vehicle and Zolpidem imaging periods for each individual cell , the duration of which was specifically increased by Zolpidem . Prior eThe frequency of calcium transients during NREM was substantially lower than during active exploration (baseline) in both vehicle and Zolpidem conditions  deep, evolutionary preserved homologous structures such as hippocampus; (b) relevant brain circuits with largely preserved natural connectivity within those circuits; (c) after systemic administration of a drug; (d) while simultaneously observing drug’s effect on animal’s behavior and physiology. While it is possible to achieve (a)-(d) by other techniques such as multi-electrode array recordings, calcium imaging is advantageous for the drug discovery settings because it has higher neuronal yield and allows investigation of drug effects in genetically identified neuronal populations We investigated the effects of Zolpidem, a commonly used treatment for insomnia, on the calcium dynamics of neurons expressing GCaMP3 calcium indicator in the hippocampus, a brain structure that is critically involved in memory, cognition and conscious experience  decreased activity of hippocampal CA1 pyramidal neurons of freely behaving mice : the ratThe suppression of calcium fluorescent signals following Zolpidem administration exceeded levels expected from decreases in locomotion . It was It is important to characterize drug effects at the level of individual neurons as studying exclusively net changes could lead to misinterpretations of the pharmacological effects. For example, a given post-drug change in net neuronal activity can be due to some minor yet consistent changes across individual cells, or because of the major changes in a minority of neurons. Furthermore, pharmacological agents can differentially influence activity of distinct neuronal subpopulations. In this case, no changes in net neuronal activity can either mean that a drug doesn’t have an effect or that a drug evokes opposite responses in different neuronal populations. These cases can be easily differentiated by studying calcium dynamics on a level of genetically identified individual cells. To demonstrate this approach, we characterized the effects of Zolpidem at the level of individual pyramidal (excitatory) CA1 hippocampal neurons, taking advantage of a selective promoter CaMKII. While the net effect of Zolpidem on calcium fluorescent signaling was predominantly suppressive, individual hippocampal pyramidal neurons in CA1 differed with respect to their responses to Zolpidem . We broain vitro literature Because the pharmacology of Zolpidem has been extensively characterized While Zolpidem predominantly lowered the frequency of calcium transients, some neurons (∼3%) responded with a marked increase in their activity  to drug discovery and to demonstrate that this approach allows for in-depth investigation of the connections between changes in the calcium signals and the physiological parameters commonly associated with the drug action. We compared calcium dynamics of individual hippocampal cells following vehicle or Zolpidem administration during NREM sleep. We selected this period because (a) Zolpidem specifically increases NREM, as was observed in current study  and prevThe NREM sleep was associated with a low rate of detected calcium transients in CA1 . This reThis study shows that it is now feasible to investigate the effects of pharmacological agents on activity of large number of genetically identified neurons in relevant brain circuits in freely behaving animals with concurrent measure of behavioral and physiological parameters. By linking molecular mechanisms, neural circuit dynamics and animal behavior, this approach has the potential to deepen our understanding of normal and abnormal brain function and how brain networks are impacted by pharmacological agents. For example, the observed suppression of hippocampal activity following Zolpidem administration provides a direct experimental evidence for a simple mechanistic explanation of adverse effects of Zolpidem on episodic memory Figure S1Histological examination of the tissue following the imaging procedures. A: A representative example of placement of the optical cannula over CA1 region of the hippocampus . Green: GCaMP3 fluorescence. Blue: DAPI staining. B: A representative example of the GCaMP3 fluorescence (green) under the optical cannula .(TIF)Click here for additional data file.Figure S2Average rate of calcium transients in all animals used in the study plotted versus average speed of the animals in each condition .(TIF)Click here for additional data file.Figure S3Distributions of normalized drug index: (post-drug event rate - post-vehicle event rate)/(post-drug event rate+post-vehicle event rate). A: The distribution of drug indices expected if Zolpidem had no effect was constructed by re-sampling, with replacement, vehicle data bins within each cells (1000 shuffles). B. The observed distribution of drug indices calculated for each cell (n = 1275). Vertical line indicates 99% confidence interval (3 s.d.) calculated from the expected distribution.(TIF)Click here for additional data file.Video S12+ transients identification applied to five representative traces shown on Representative videos of imaging sessions with vehicle and Zolpidem illustrate Ca. Ca2+ transients were identified by searching each trace for local maxima that had peak amplitude more than two standard deviations  from the baseline ; an occurrence of a calcium transient is indicated as a tick mark.(ZIP)Click here for additional data file.Video S2Representative movies of imaging sessions with vehicle and Zolpidem (in the absence of ambulatory movements in both conditions). The two movies  display relative change in fluorescence (ΔF′(t)/F′0; shown on 5% scale and sped-up 10 times) recorded in the same 640×570 µm2 imaging field.(ZIP)Click here for additional data file.


Factors influencing differential responses of prostate tumors to androgen receptor (AR) axis-directed therapeutics are poorly understood, and predictors of treatment efficacy are needed. We hypothesized that the efficacy of inhibiting DHT ligand synthesis would associate with intra-tumoral androgen ratios indicative of relative dependence on DHT-mediated growth.We characterized two androgen-sensitive prostate cancer xenograft models after androgen suppression by castration in combination with the SRD5A inhibitor, dutasteride, as well as a panel of castration resistant metastases obtained via rapid autopsy.In LuCaP35 tumors  dutasteride suppressed DHT to 0.02 ng/gm and prolonged survival vs. castration alone . In LuCaP96 tumors (T:DHT 10∶1), survival was not improved despite similar DHT reduction (0.02 ng/gm). LuCaP35 demonstrated higher expression of steroid biosynthetic enzymes maintaining DHT levels , reconstitution of intra-tumoral DHT (to ∼30% of untreated tumors), and ∼2 fold increased expression of full length AR. In contrast, LuCaP96 demonstrated higher levels of steroid catabolizing enzymes , persistent suppression of intra-tumoral DHT, and 6–8 fold induction of full length AR and the ligand independent V7 AR splice variant. Human metastases demonstrated bio-active androgen levels and AR full length and AR splice-variant expression consistent with the range observed in xenografts.Intrinsic differences in basal steroidogenesis, as well as variable expression of full length and splice-variant AR, associate with response and resistance to pre-receptor AR ligand suppression. Expression of steroidogenic enzymes and AR isoforms may serve as potential biomarkers of sensitivity to potent AR-axis inhibition and should be validated in additional models. The clinical responses to new potent AR antagonists and novel steroid synthesis inhibitors, coupled with evidence of intracrine androgen biosynthesis in prostate cancer suggest that concepts of maximal androgen blockade should be revisited and refined. A recent study designed to target multiple steps in androgen biosynthesis in men failing ADT  dutasteride) produced substantial PSA declines in most patients and a median response duration of 20 months. In this study, we sought to determine tumor-specific characteristics that influence response of prostate cancers to inhibition of pre-receptor AR signaling, using androgen deprivation combined with the dual SRD5A inhibitor dutasteride. T and DHT are biologically active high-affinity AR ligands. However, between 3–10 fold higher concentrations of T are required to exert the same AR-mediated transcriptional effects of DHT. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All experiments involving animals were performed in accordance with protocols approved by the Fred Hutchinson Center Institutional Animal Care Use Committee (file 1775). All surgery was performed under isofluorane anesthesia, and all efforts were made to minimize suffering. The LuCaP35 and LuCaP96 lines are patient derived xenografts that were established as part of the University of Washington tissue bank as previously described 3 tumor pieces. When tumors reached an average of 300 mm3, mice  were castrated (Cx) and randomized to treatment with vehicle or dutasteride (Dut) therapy over 8 weeks. Dutasteride was administered (PEG monolaurate (Sigma 460133)/1% Tween 80 (Sigma P4780)) by oral gavage 5 days per week in a volume of 200µl. Tumor volume was determined by the following formula (long and short axis lengths in mm): long×(Short∧2)/2. Tumors from a subset of mice in each cohort were harvested at early time points of treatment . When tumors reached approximately 750–1000 mg in size, the animals were euthanized according to institutional protocol and the xenografts harvested and flash frozen for determination of tissue androgens and extraction of total RNA. The average number of days mice in the Cx+Dut groups had been off dutasteride treatment at the time tumors were resected was 211 days for LuCaP35, and 96 days for LuCaP96. Dutasteride was generously provided by GlaxoSmithKline.Male C.B-17 SCID mice  were implanted subcutaneously with 30 mmAll procedures involving human subjects were approved by the Institutional Review Board of the University of Washington Medical Center, and all subjects signed written informed consent. Patients with metastatic prostate cancer underwent rapid autopsies under the aegis of the University of Washington Prostate Cancer Donor Autopsy Program as previously described Androgen levels in xenograft tissues were determined by mass spectrometry (MS) using methods we have described. Samples were used for RNA isolation, cDNA synthesis and quantitative RT-PCR using methods and primers we have previously described. 3) was determined via Kaplan Meier analysis with comparison of curves using the Mantel-Haenszel logrank test. To predict the approximate sample size in each treatment group that might be necessary to detect a statistically significant effect of castration plus dutasteride versus castration alone, we utilized complete case resampling for a range of sample sizes n = 10, 20, …, 200 over 500 bootstrap replicates. For each sample size and bootstrap replicate, we fit a Cox proportional hazards model and recorded the estimated hazard ratio and p-value.To evaluate differences in tumor volume trajectory, we first determined change points in the tumor growth curves using t-tests to sequentially compare tumor volumes every two days. After segmenting the curves, we applied a linear mixed model to quantify the treatment effect on tumor growth for each linear period by regressing log tumor volume on day of measurement, on treatment group, and on an interaction between day and treatment while accounting for variability in initial tumor volume across mice. Progression free survival in castration vs. castration + dutasteride treated mice (defined as tumor size <750 mmΔΔCt). The raw dCT’s, ddCT’s and fold changes are presented in the Supplemental data.For analysis of qRT-PCR data, the mean cycle threshold (Ct) for each gene was normalized to expression of the housekeeping gene RPL13A in the same sample (delta Ct). The mean expression of RPL13A was 15.6+/−0.5 (mean +/− SD) and 14.1+/−0.8 in LuCaP35 and LuCap96 tumors respectively. Welch’s two sample t-tests were used to compare differences in androgen levels and differences in mean delta Ct’s for each gene between treatment groups without correction for multiple testing. P values<0.05 were considered significant. The fold change was calculated by the delta-delta Ct method (fold = 2Table S1).To evaluate tumor characteristics associated with response and resistance to AR pathway-directed therapy, we first determined intratumoral androgens and steroidogenic gene expression in two prostate cancer patient derived xenografts, LuCaP35 and LuCaP96. These lines express AR and PSA, . While many steroidogenic genes were expressed in LuCaP35 within one order of magnitude of their expression in LuCaP96 (Table S2). Since these tumors were propagated in genetically-identical murine hosts, these data identify intrinsic tumor-specific variation in androgen metabolic programs culminating in marked intratumoral differences in AR ligand concentrations.To identify mechanisms contributing to basal differences in intra-tumoral T and DHT concentrations, we evaluated tumor-specific expression of transcripts encoding the primary enzymes involved in steroid synthesis and degradation  at the time of enrollment ; however, this may reflect decreased power associated with the post-hoc sub-classification of the groups.To further examine tumor characteristics associated with response to pre-receptor AR pathway inhibition, we determined whether response to treatment was related to tumor size at initiation of therapy. Although the study was designed to begin treatment at a tumor volume of ∼300 mm3 at enrollment (3 at enrollment (Inspection of tumor volume curves suggested a suppressive effect of dutasteride was maintained even after discontinuation of therapy for tumors <250 mmTable S1).To determine whether the differential impact of SRD5A inhibition on tumor growth reflected a difference in suppression of intratumoral androgens, we measured T and DHT levels in tumors resected after treatment. In both LuCaP35 and LuCaP96 tumor types, mean tumor DHT levels measured at 3–21 days (indicated by two-headed arrows in Table S1). Of note, the half-life of dutasteride in mice is less than 2 days, Interestingly, while T levels in LuCaP35 tumors remained suppressed in either treatment group . Collectively, these data show that dutasteride effectively inhibited DHT production in both tumor types, and that LuCaP35 tumor growth is susceptible to the enhanced pre-receptor suppression of DHT achieved with the combination of castration plus SRD5A inhibition, whereas LuCaP96 is not.The tendency toward reconstitution of DHT levels in LuCaP35 and of T in LuCaP96 is consistent with the intrinsic variation in androgen metabolic programs favoring DHT vs. T identified in the untreated tumors (FL), and as recently reported, by increased expression of ARsv that lack the LBD and consequently confer constitutive ligand-independent AR activity. Differences in ARFL and ARsv levels may contribute to the variation in tumor growth responses following pre-receptor ligand suppression. Therefore, we evaluated the expression of ARFL and the prevalent ARsv identified in human CRPC samples, ARV7 (which encodes the same protein as ARV3) in LuCaP35 and LuCaP96 tumors recurring after ADT and dutasteride Castration resistant prostate tumors are frequently characterized by increased expression of full-length AR . In contrast, while LuCaP96 tumors recurring after castration alone demonstrated a similar 1.9-fold increased expression of ARFL, they also showed a substantial 10-fold increase in the expression of ARV7 . However, significant differences were generally not observed in recurrent tumors resected at late time points, consistent with the increase in AR expression (Table S3) and reconstitution of tumor androgen levels observed in these samples. Interestingly, expression of FKBP5 remained quite low in recurrent LuCap96 tumors despite the marked increase in ARV7, suggesting ARV7 does not necessarily completely recapitulate the transcriptional activity of full length AR.Compared to tumors resected from intact mice, LuCaP35 tumors recurring after castration or after castration plus dutasteride demonstrated a 2.0-fold increase in expression of ARV7 in LuCaP96, we did not observe a parallel increase in the absolute expression of genes previously suggested to constitute the variant transcriptome . (13–16) However the induction of ARV7 in LuCap96 was associated with a significant shift in the correlation of its expression with AKT. Whereas the expression of ARV7 and AKT was correlated in LuCaP35 tumors under all treatment conditions , expression of these genes only became correlated in LuCaP96 tumors when ARV7 was induced after treatment .Despite the increased expression of ARsv may account for the differential impact of pre-receptor androgen suppression on tumor-growth inhibition, with the marked rise in ARV7 observed in LuCaP96 rendering it correspondingly less sensitive to ligand suppression by dutasteride. Of note, consistent expression of the ARv567 variant also reported in human CRPC tissues was not detected in these xenografts (data not shown) These data suggest that tumor-specific differences in the induction of full-length AR and ARFL (as represented by LuCaP35), whereas another is characterized by robust induction of ARsv, rather than metabolic adaptations directed at increased DHT synthesis (as represented by LuCaP96). To determine whether these observations are relevant in context of in vivo human prostate cancer, we compared relative levels of T, DHT and AR expression in tumor metastases directly obtained from men with CRPC. Given the 5–10 fold higher potency of DHT for engaging and activating AR relative to T, sv are not available, AR immunostaining was performed using antibodies recognizing the N-terminus (ARN+) or C-terminus (ARC+) of the AR protein to identify tumors expressing predominantly ARFL , and those with C-terminal truncated ARsv  The studies of LuCaP35 and LuCaP96 xenografts support the existence of at least two subtypes of CRPC. One type is reliant on androgen biosynthesis and intratumoral maintenance of the potent AR ligand DHT, with modest upregulation of ligand-activated AR). Moreover, patients 1 and 3 whose metastases were characterized by a higher androgenicity index with a relatively larger contribution of DHT (similar to castration recurrent LuCaP35) demonstrated primarily ARN+ and ARC+ staining, consistent with detection of ARFL, and were consistently PSA positive  compared to LuCaP35 (0.40±0.56 ng/gm), as emerging data suggest that induction of AR variants reflects a dynamic response to ligand inhibition. V7 is not markedly induced by suppression of DHT to 0.02±0.04 ng/gm in LuCaP35 tumors treated with castration plus dutasteride, suggesting tumors may also possess intrinsic differences in the propensity for generation of ARSV. Our data demonstrating significant levels of intratumoral T in LuCaP96 are in contrast to the study of Chang et al, which suggested that the route to intratumoral DHT synthesis can bypass T. Our findings indicate that this is not the case in every instance, and emphasize the range of tumor resistance mechanisms likely to be encountered in CRPC tumors Factors responsible for the preferential induction of steroidogenesis versus induction of ARSV, as has been observed in a subset of hormone naïve primary prostate cancers, The impact of DHT suppression on tumor growth inhibition in LuCaP35 was influenced by tumor size at initiation of therapy, with smaller tumors demonstrating a more profound suppression of tumor growth which also appeared to be durable after discontinuation of therapy, but larger tumors demonstrating suppression of tumor growth only while actively receiving treatment. These data suggest that even in tumors that are sensitive to androgen suppression, the potential efficacy associated with ligand synthesis inhibitors may be more pronounced and/or more durable if initiated earlier at a lower volume of disease. These data also provide a hypothesis for results of the PCPT and REDUCE clinical trials using SRD5A inhibitors for prostate cancer chemoprevention. in situ metastases demonstrate a patient-specific range of tumor androgen levels and associated differences in AR expression similar to that observed in LuCaP35 and LuCaP96 xenografts. Specifically, human metastases characterized by higher tissue androgen index demonstrated evidence of full length AR expression (similar to LuCaP35), whereas metastases with a relatively lower tissue androgen index demonstrated loss of C terminal AR staining, consistent with expression of ligand independent AR variants (as observed in LuCaP96). These data suggest that our findings regarding response and resistance to treatment strategies targeting pre-receptor androgen pathway suppression in the LuCaP35 and LuCaP96 xenograft models are likely to be relevant to the clinical spectrum of disease encountered in patients with CRPC.Notably, direct examination of human CRPC tumors showed that FL or ARSV as potential mechanisms of resistance in tumors recurring after treatment with abiraterone. SV, at a magnitude that may be sufficient to impede sensitivity to additional androgen ablation, can occur following castration alone, as observed in LuCaP96 tumors. Clearly, these represent prostate tumors for which agents acting through mechanisms independent of ligand binding have a higher likelihood of efficacy than agents targeting ligand synthesis or the LBD. Consistent with this hypothesis, recent data demonstrate that patients with detectable ARSV in circulating tumor cells have a poor response to the LBD antagonist enzalutamide. Importantly, the observations reported in the present study may be relevant to optimizing first-line treatment strategies with novel ligand-synthesis and AR targeted agents such as abiraterone and enzalutamide. While the clinical responses to abiraterone and enzalutamide have been impressive, not all patients respond, the duration of response has been variable, and mechanisms of resistance are not well understood. SV and steroidogenic enzyme expression as biomarkers for treatment success. Collectively, however, these studies suggest that intrinsic differences in basal steroidogenesis and expression of ligand-independent ARSV may associate with response and resistance to pre-receptor suppression of AR ligands, and may directly underlie the variable clinical efficacy observed in clinical studies of abiraterone and enzalutamide in men with CRPC. Recent data demonstrating that enzalutamide, which targets the AR LBD, had decreased efficacy against tumors expressing LBD-deleted ARSV, suggests these tumors may require agents targeting the AR N terminus, and is a critical subject of ongoing research. FL and ARSV as potential biomarkers of response and resistance requires assessment in ongoing clinical studies employing molecular evaluation of tumor tissue from men with CRPC.An important limitation of this study is that only two xenograft models were characterized, and evaluation of further models will be required to establish the validity of ARFigure S1The Androgen Synthetic Pathway. The schema depicts the enzymatic steps in the conversion of cholesterol to androgens. Steroid intermediates are given in the boxes. Steroidogenic enzymes mediating each step are given next to the arrow denoting the direction of the conversion. After being generated from cholesterol by CYP11A C21 steroids are converted to the C19 adrenal androgens DHEA by the sequential hydroxylase and lyase activity of CYP17A. DHEA is acted on by HSD3B to form androstenedione (AED), which in the classical pathway is then acted on by HSD17B3 (or AKR1C3) to form testosterone, which is converted to DHT via SRD5A. In the 5-alpha-Androstanedione pathway AED is converted first by SRD5A to 5α-Androstanedione and then by HSD17B3 (or AKR1C3) to DHT. In the backdoor pathway the progestin intermediates are acted on first by the activity of SRD5A and the reductive activity of AKR1C2 prior to the lyase activity of CYP17A to form androsterone (not shown). Androsterone is then acted on by HSD17B3 (or AKR1C3) and must undergo an oxidative step mediated by RL-HSD (or others) to generate DHT.(TIF)Click here for additional data file.Figure S2Expression of steroidogenic genes in LuCap35 and LuCaP96 prostate cancer xenografts. Relative expression of the indicated steroidogenic genes in LuCaP35 and LuCaP96 in tumors grown in intact mice  and in tumors re-growing after castration (B) or castration+8 weeks dutasteride (C). Transcript levels were measured by qRT-PCR and normalized to the housekeeping gene RPL13A within each sample to yield the delta cycle threshold (dCT). For each gene the relative difference in mean expression between LuCaP35 and LuCaP96 was calculated using the delta dCt method (fold change = 2∧ddCtT). Genes differentially expressed in LuCaP35 vs. LuCaP96 within one order of magnitude are indicated within the gray lines. Significant differences  are indicated by black circles and colored triangles; white circles indicate genes that were not significantly different between LuCaP35 and LuCaP96. Upward red triangles indicate highly differentially expressed genes leading to increased T (AKR1C3) and increased DHT levels . Downward green triangles indicate highly differentially expressed genes mediating DHT catabolism .(TIF)Click here for additional data file.Table S1Androgen levels in LuCaP35 and LuCaP96 tumors after pre-receptor androgen suppression with castration or castration + dutasteride.(TIF)Click here for additional data file.Table S2Fold difference in mean transcript expression of steroidogenic genes between LuCaP35 and LuCaP96 tumors grown in intact mice.(TIF)Click here for additional data file.Table S3Expression of AR and AR-regulated genes in LuCaP35 and LuCaP96 tumors after pre-receptor androgen suppression with castration or castration + dutasteride.(TIF)Click here for additional data file.