Motor and cognitive functions depend on the coordinated interactions between dopamine (DA) and acetylcholine (ACh) at striatal synapses. Increased ACh availability was assumed to accompany DA deficiency based on the outcome of pharmacological treatments and measurements in animals that were critically depleted of DA. Using Slc6a3 DTR/+ diphtheria-toxin-sensitive mice, we demonstrate that a progressive and L-dopa-responsive DA deficiency reduces ACh availability and the transcription of hyperpolarization-activated cation (HCN) channels that encode the spike timing of ACh-releasing tonically active striatal interneurons (ChIs). Although the production and release of ACh and DA are reduced, the preponderance of ACh over DA contributes to the motor deficit. The increase in striatal ACh relative to DA is heightened via D1-type DA receptors that activate ChIs in response to DA release from residual axons. These results suggest that stabilizing the expression of HCN channels may improve ACh-DA reciprocity and motor function in Parkinson's disease (PD).

Phenomics, which ideally involves in-depth phenotyping at the whole-organism scale, may enhance our functional understanding of genetic variation. Here, we demonstrate methods to profile hundreds of phenotypic measures comprised of morphological and densitometric traits at a large number of sites within the axial skeleton of adult zebrafish. We show the potential for vertebral patterns to confer heightened sensitivity, with similar specificity, in discriminating mutant populations compared to analyzing individual vertebrae in isolation. We identify phenotypes associated with human brittle bone disease and thyroid stimulating hormone receptor hyperactivity. Finally, we develop allometric models and show their potential to aid in the discrimination of mutant phenotypes masked by alterations in growth. Our studies demonstrate virtues of deep phenotyping in a spatially distributed organ system. Analyzing phenotypic patterns may increase productivity in genetic screens, and facilitate the study of genetic variants associated with smaller effect sizes, such as those that underlie complex diseases.

Phenomics, which ideally involves in-depth phenotyping at the whole-organism scale, may enhance our functional understanding of genetic variation. Here, we demonstrate methods to profile hundreds of phenotypic measures comprised of morphological and densitometric traits at a large number of sites within the axial skeleton of adult zebrafish. We show the potential for vertebral patterns to confer heightened sensitivity, with similar specificity, in discriminating mutant populations compared to analyzing individual vertebrae in isolation. We identify phenotypes associated with human brittle bone disease and thyroid stimulating hormone receptor hyperactivity. Finally, we develop allometric models and show their potential to aid in the discrimination of mutant phenotypes masked by alterations in growth. Our studies demonstrate virtues of deep phenotyping in a spatially distributed organ system. Analyzing phenotypic patterns may increase productivity in genetic screens, and facilitate the study of genetic variants associated with smaller effect sizes, such as those that underlie complex diseases.

Phenomics, which ideally involves in-depth phenotyping at the whole-organism scale, may enhance our functional understanding of genetic variation. Here, we demonstrate methods to profile hundreds of measures comprised of morphological and densitometric traits from a large number sites in the axial skeleton of adult zebrafish. We show the potential for vertebral patterns to confer heightened sensitivity, with similar specificity, in discriminating mutant populations compared to analyzing individual vertebrae in isolation. We identify phenotypes associated with human brittle bone disease and thyroid stimulating hormone receptor hyperactivity. Finally, we develop allometric models and show their potential to aid in the discrimination of mutant phenotypes masked by alterations in growth. Our studies demonstrate virtues of deep phenotyping in a spatially distributed organ. Analyzing phenotypic patterns may increase productivity in genetic screens, and could facilitate the study of genetic variants associated with smaller effect sizes, such as those that underlie complex diseases. Using a high-throughput automated synchrotron-based tomographic microscopy system, Mader et al. [7] quantified 22 different measurements in the mouse femur. In zebrafish, Pardo-Martin et al. [8] used automated sample handling and optical projection tomography to acquire high-dimensional phenotypic profiles (~200 measurements) in the craniofacial cartilage of early larvae, representing one of the most ambitious approachesto perform large-scale phenotyping in the skeleton to date. Yet, even in this analysis, traits were derived from only 9 skeletal elements. Further, this method is not readily extendable to bones outside of the craniofacial skeleton, or to adults. Finally, while both the mouse and zebrafish spine are amenable to whole-body microCT imaging [9, 10], indepth phenotyping is usually limited to a few vertebral bodies [11]. In this context, methods to perform in-depth phenotyping in a large number of bones represents a unique class of problems that has not been adequately addressed.

Corticostriatal signaling participates in sensitized responses to drugs of abuse, where short-term increases in dopamine availability provoke persistent, yet reversible, changes in glutamate release. Prior studies in mice show that amphetamine withdrawal promotes a chronic presynaptic depression in glutamate release, whereas an amphetamine challenge reverses this depression by potentiating corticostriatal activity in direct pathway medium spiny neurons. This synaptic plasticity promotes corticostriatal activity and locomotor sensitization through upstream changes in the activity of tonically active cholinergic interneurons (ChIs). We used a model of operant drug-taking behaviors, in which mice self-administered amphetamine through an in-dwelling catheter. Mice acquired amphetamine self-administration under fixed and increasing schedules of reinforcement. Following a period of abstinence, we determined whether nicotinic acetylcholine receptors modified drug-seeking behavior and associated alterations in ChI firing and corticostriatal activity. Mice responding to conditioned reinforcement showed reduced ChI and corticostriatal activity ex vivo, which paradoxically increased following an amphetamine challenge. Nicotine, in a concentration that increases Ca2+ influx and desensitizes α4β2*-type nicotinic receptors, reduced amphetamine-seeking behaviors following abstinence and amphetamine-induced locomotor sensitization. Nicotine blocked the depression of ChI firing and corticostriatal activity and the potentiating response to an amphetamine challenge. Together, these results demonstrate that nicotine reduces reward-associated behaviors following repeated amphetamine and modifies the changes in ChIs firing and corticostriatal activity. By returning glutamatergic activity in amphetamine self-administering mice to a more stable and normalized state, nicotine limits the depression of striatal activity in withdrawal and the increase in activity following abstinence and a subsequent drug challenge.

Phenomics, which ideally involves in-depth phenotyping at the whole-organism scale, may enhance our functional understanding of genetic variation. Here, we demonstrate methods to profile hundreds of phenotypic measures comprised of morphological and densitometric traits at a large number of sites within the axial skeleton of adult zebrafish. We show the potential for vertebral patterns to confer heightened sensitivity, with similar specificity, in discriminating mutant populations compared to analyzing individual vertebrae in isolation. We identify phenotypes associated with human brittle bone disease and thyroid stimulating hormone receptor hyperactivity. Finally, we develop allometric models and show their potential to aid in the discrimination of mutant phenotypes masked by alterations in growth. Our studies demonstrate virtues of deep phenotyping in a spatially distributed organ system. Analyzing phenotypic patterns may increase productivity in genetic screens, and facilitate the study of genetic variants associated with smaller effect sizes, such as those that underlie complex diseases.

No abstract

Introduction: Oncogenic WNT/β-catenin signaling and activating mutations that increase stability and transcriptional activity of β-catenin are among the most frequent lesions throughout all main types of cancer (Morin et al. 1997; Rubinfeld et al. 1997). Results: Strikingly, pan-cancer analysis of activating mutations of β-catenin in patient samples and global proteomic analysis of human cancer cell lines revealed that B-cell malignancies are exempt from oncogenic activation of β-catenin. While nuclear accumulation of β-catenin was observed in >80% of cancer samples studied, nuclear β-catenin was consistently absent in normal and malignant B-lymphoid cells. To model the effects of inducible β-catenin activation in B-ALL, pre-B cells from Ctnnb1 ex3fl/+ mice were transformed with BCR-ABL1 or NRAS G12D oncogenes. Cre-mediated excision of exon 3 removes GSK3β and CK1 phosphorylation sites (S 33, S 37, T 41 and T 45) required for β-catenin degradation, therefore resulting in accumulation of β-catenin. Stabilization of β-catenin rapidly impaired competitive fitness of B-ALL cells, abolished colony forming ability and induced cell cycle arrest. Likewise, expression of constitutively active form of β-catenin in patient derived B-ALL cells (n=6) or B-cell lymphoma (n=4) compromised proliferation, clonal fitness, and induced cell death. Interestingly, activation of β-catenin signaling in myeloid leukemia (n=4) accelerated proliferation. Corroborating the lineage specific effects of β-catenin, CEBPα-mediated reprogramming of B-ALL cells into myeloid lineage leukemia cells, reversed the deleterious effect of inducible β-catenin-activation. In epithelial cell types, β-catenin/TCF complexes drive transcriptional activation of MYC (He et. al.1998). In striking contrast to epithelial cells, however, our transcriptomic analysis showed that inducible activation of β-catenin in B-cell malignancies resulted in transcriptional repression of Myc and its target genes. Surprisingly, proximity-based labeling and interactome studies in B-ALL cells revealed Ikzf1 and Ikzf3 as top-ranking interaction partners for β-catenin. Furthermore, several components of the Nucleosome Remodeling and Deacetylase (NuRD) complex including Mta1/2, Gatad2a/b, Chd4, Hdac1/2 were among the most significantly enriched proteins within the β-catenin interactome. Interaction of Ikzf1 and Ikzf3 with NuRD complex induced loss of histone H3 lysine 27 acetylation (H3K27Ac) and suppressed Myc expression. To test whether repressive complexes of β-catenin with Ikzf1, Ikzf3:NuRD subvert proliferation and survival by transcriptional repression of Myc, we introduced Cas9-RNP for genetic deletion of Ikzf1 and Ikzf3 in B-ALL cells. Interestingly, deletion of both Ikzf1 and Ikzf3 was required to restore clonal fitness, proliferation and Myc-driven transcriptional programs. Conversely, genetic deletion of β-catenin was sufficient to abrogate Ikzf1/Ikzf3-mediated tumor suppression and transcriptional repression of Myc. Together, these results suggest that engagement of b-catenin set the threshold for Ikzf1/Ikzf3-mediated tumor suppression in B-ALL and mature B-cell malignancies. Together with B-lineage-specific expression of Ikzf1 and Ikzf3, these findings provide a mechanistic explanation as to why activating lesions of β-catenin are not detected in B-cell malignancies. Therapeutic implication: To leverage the unique sensitivity of B-cell malignancies to β-catenin-activation, we tested pharmacological activation of β-catenin based on small molecule-inhibition of GSK3β, a central negative regulator of β-catenin. Testing six different GSK3β inhibitors, we identified LY2090314 as the most potent inhibitor in killing B-ALL cells and suppressing Myc (IC50=4.5 nM). Treatment of NSG mice bearing patient derived B-ALL xenografts with LY2090314 substantially reduced leukemia burden and significantly extended overall survival compared to vehicle treated mice (n=9, P= 6.5E-05). These findings suggest that β-catenin activation is a unique vulnerability and small molecule GSK3β inhibition represents a novel opportunity to overcome drug-resistance in refractory B-ALL. Disclosures No relevant conflicts of interest to declare.