Probes for INS

Molecular identification and characterization of fear controlling circuitries is a promising path towards developing targeted treatments of fear-related disorders. Three-color in situ hybridization analysis was used to determine whether somatostatin (Sst), neurotensin (Nts), corticotropin releasing factor (Crf), tachykinin 2 (Tac2), protein kinase c delta (Prkcd), and dopamine receptor 2 (Drd2) mRNA co-localize in male mouse amygdala neurons. Expression and co-localization was examined across capsular (CeC), lateral (CeL), and medial (CeM) compartments of the central amygdala. The greatest expression of Prkcd and Drd2 were found in CeC and CeL. Crf was expressed primarily in CeL while Sst, Nts, and Tac2 expressing neurons were distributed between CeL and CeM. High levels of co-localization were identified between Sst, Nts, Crf, and Tac2 within the CeL while little co-localization was detected between any mRNAs within the CeM. These findings provide a more detailed understanding of the molecular mechanisms that regulate the development and maintenance of fear and anxiety behaviors.

Significance Statement Functional and behavioral analysis of central amygdala microcircuits has yielded significant insights into the role of this nucleus in fear and anxiety related behaviors. However, precise molecular and locational description of examined populations is lacking. This publication provides a quantified regionally precise description of the expression and co-expression of six frequently examined central amygdala population markers. Most revealing, within the most commonly examined region, the posterior CeL, four of these markers are extensively co-expressed suggesting the potential for experimental redundancy. This data clarifies circuit interaction and function and will increase relevance and precision of future cell-type specific reports.

Hair growth disorders often carry a major psychological burden. Therefore, more effective human hair growth-modulatory agents urgently need to be developed. Here, we used the hypertrichosis-inducing immunosuppressant, Cyclosporine A (CsA), as a lead compound to identify new hair growth-promoting molecular targets. Through microarray analysis we identified the Wnt inhibitor, secreted frizzled related protein 1 (SFRP1), as being down-regulated in the dermal papilla (DP) of CsA-treated human scalp hair follicles (HFs) ex vivo. Therefore, we further investigated the function of SFRP1 using a pharmacological approach and found that SFRP1 regulates intrafollicular canonical Wnt/β-catenin activity through inhibition of Wnt ligands in the human hair bulb. Conversely, inhibiting SFRP1 activity through the SFRP1 antagonist, WAY-316606, enhanced hair shaft production, hair shaft keratin expression, and inhibited spontaneous HF regression (catagen) ex vivo. Collectively, these data (a) identify Wnt signalling as a novel, non-immune-inhibitory CsA target; (b) introduce SFRP1 as a physiologically important regulator of canonical β-catenin activity in a human (mini-)organ; and (c) demonstrate WAY-316606 to be a promising new promoter of human hair growth. Since inhibiting SFRP1 only facilitates Wnt signalling through ligands that are already present, this 'ligand-limited' therapeutic strategy for promoting human hair growth may circumvent potential oncological risks associated with chronic Wnt over-activation.

Endogenous dynorphin signaling via the kappa-opioid receptor (KOR) in the nucleus accumbens (NAcc) powerfully mediates negative affective states and stress reactivity. Excitatory inputs from the hippocampus and amygdala play a fundamental role in shaping the activity of both NAcc D1 and D2 MSNs, which encode positive and negative motivational valences, respectively. However, a circuit-based mechanism by which KOR modulation of excitation-inhibition balance modifies D1 and D2 MSN activity is lacking. Here, we provide a comprehensive synaptic framework wherein presynaptic KOR inhibition decreases the excitatory drive of D1 MSN activity by the amygdala, but not the hippocampus. Conversely, presynaptic inhibition by KORs of inhibitory synapses on D2 MSNs enhances integration of excitatory drive by the amygdala and hippocampus. In conclusion, we describe a circuit-based mechanism showing differential gating of afferent control of D1 and D2 MSN activity by KORs in a pathway-specific manner.

Behavioral and molecular characterization of cell-type-specific populations governing fear learning and behavior is a promising avenue for the rational identification of potential therapeutics for fear-related disorders. Examining cell-type-specific changes in neuronal translation following fear learning allows for targeted pharmacological intervention during fear extinction learning, mirroring possible treatment strategies in humans. Here we identify the central amygdala (CeA) Drd2-expressing population as a novel fear-supporting neuronal population that is molecularly distinct from other, previously identified, fear-supporting CeA populations. Sequencing of actively translating transcripts of Drd2 neurons using translating ribosome affinity purification (TRAP) technology identifies mRNAs that are differentially regulated following fear learning. Differentially expressed transcripts with potentially targetable gene products include Npy5r, Rxrg, Adora2a, Sst5r, Fgf3, Erbb4, Fkbp14, Dlk1, and Ssh3. Direct pharmacological manipulation of NPY5R, RXR, and ADORA2A confirms the importance of this cellpopulation and these cell-type-specific receptors in fear behavior. Furthermore, these findings validate the use of functionally identified specific cell populations to predict novel pharmacological targets for the modulation of emotional learning.

Spatiotemporal control of Wnt signaling is essential for the development and homeostasis of many tissues. The transmembrane E3 ubiquitin ligases ZNRF3 (zinc and ring finger 3) and RNF43 (ring finger protein 43) antagonize Wnt signaling by promoting degradation of frizzled receptors. ZNRF3 and RNF43 are frequently inactivated in human cancer, but the molecular and therapeutic implications remain unclear. Here, we demonstrate that adrenocortical-specific loss of ZNRF3, but not RNF43, results in adrenal hyperplasia that depends on Porcupine-mediated Wnt ligand secretion. Furthermore, we discovered a Wnt/β-catenin signaling gradient in the adrenal cortex that is disrupted upon loss of ZNRF3. Unlike β-catenin gain-of-function models, which induce high Wnt/β-catenin activation and expansion of the peripheral cortex, ZNRF3 loss triggers activation of moderate-level Wnt/β-catenin signaling that drives proliferative expansion of only the histologically and functionally distinct inner cortex. Genetically reducing β-catenin dosage significantly reverses the ZNRF3-deficient phenotype. Thus, homeostatic maintenance of the adrenal cortex is dependent on varying levels of Wnt/β-catenin activation, which is regulated by ZNRF3.

In mice, implantation always occurs towards the antimesometrial side of the uterus, while the placenta develops at the mesometrial side. What determines this particular orientation of the implanting blastocyst remains unclear. Uterine glands are critical for implantation and pregnancy. In this study, we showed that uterine gland development and active Wnt signalling activity is limited to the antimesometrial side of the uterus. Dkk2, a known antagonist of Wnt signalling, is only present at the mesometrial side of the uterus. Imaging of whole uterus, thick uterine sections (100-1000μm), and individual glands revealed that uterine glands are simple tubes with branches that are directly connected to the luminal epithelium and are only present towards the antimesometrial side of the uterus. By developing a unique mouse model targeting the uterine epithelium, we demonstrated that Wnt/β-catenin signaling is essential for prepubertal gland formation and normal implantation, but dispensable for postpartum gland development and regeneration. Our results for the first time have provided a probable explanation for the antimesometrial bias for implantation.

Basolateral amygdala (BLA) principal cells are capable of driving and antagonizing behaviors of opposing valence. BLA neurons project to the central amygdala (CeA), which also participates in negative and positive behaviors. However, the CeA has primarily been studied as the site for negative behaviors, and the causal role for CeA circuits underlying appetitive behaviors is poorly understood. Here, we identify several genetically distinct populations of CeA neurons that mediate appetitive behaviors and dissect the BLA-to-CeA circuit for appetitive behaviors. Protein phosphatase 1 regulatory subunit 1B+ BLA pyramidal neurons to dopamine receptor 1+ CeA neurons define a pathway for promoting appetitive behaviors, while R-spondin 2+ BLA pyramidal neurons to dopamine receptor 2+ CeA neurons define a pathway for suppressing appetitive behaviors. These data reveal genetically defined neural circuits in the amygdala that promote and suppress appetitive behaviors analogous to the direct and indirect pathways of the basal ganglia.

Despite recent efforts to dissect the inter-tumor heterogeneity of pancreatic ductal adenocarcinoma (PDAC) by determining prognosis-predictive gene expression signatures for specific subtypes, their functional differences remain elusive. Here, we established a pancreatic tumor organoid library encompassing 39 patient-derived PDACs and identified 3 functional subtypes based on their stem cell niche factor dependencies on Wnt and R-spondin. A Wnt-non-producing subtype required Wnt from cancer-associated fibroblasts, whereas a Wnt-producing subtype autonomously secreted Wnt ligands and an R-spondin-independent subtype grew in the absence of Wnt and R-spondin. Transcriptome analysis of PDAC organoids revealed gene-expression signatures that associated Wnt niche subtypes with GATA6-dependent gene expression subtypes, which were functionally supported by genetic perturbation of GATA6. Furthermore, CRISPR-Cas9-based genome editing of PDAC driver genes (KRAS, CDKN2A, SMAD4, and TP53) demonstrated non-genetic acquisition of Wnt niche independence during pancreas tumorigenesis. Collectively, our results reveal functional heterogeneity of Wnt niche independency in PDAC that is non-genetically formed through tumor progression.

The oral mucosa is one of the most rapidly dividing tissues in the body and serves as a barrier to physical and chemical insults from mastication, food, and microorganisms. Breakdown of this barrier can lead to significant morbidity and potentially life-threatening infections for patients. Determining the identity and organization of oral epithelial progenitor cells (OEPCs) is therefore paramount to understanding their roles in homeostasis and disease. Using lineage tracing and label retention experiments, we show that rapidly dividing OEPCs are located broadly within the basal layer of the mucosa throughout the oral cavity. Quantitative clonal analysis demonstrated that OEPCs undergo population-asymmetrical divisions following neutral drift dynamics and that they respond to chemotherapy-induced damage by altering daughter cell fates. Finally, using single-cell RNA-seq, we establish the basal layer population structure and propose a model that defines the organization of cells within the basal layer.