The liver can substantially regenerate after injury, with both main epithelial cell types, hepatocytes and biliary epithelial cells (BECs), playing important roles in parenchymal regeneration. Beyond metabolic functions, BECs exhibit substantial plasticity and in some contexts can drive hepatic repopulation. Here, we performed single-cell RNA sequencing to examine BEC and hepatocyte heterogeneity during homeostasisand after injury. Instead of evidence for a transcriptionally defined progenitor-like BEC cell, we found significant homeostatic BEC heterogeneity that reflects fluctuating activation of a YAP-dependent program. This transcriptional signature defines a dynamic cellular state during homeostasis and is highly responsive to injury. YAP signaling is induced by physiological bile acids (BAs), required for BEC survival in response to BA exposure, and is necessary for hepatocyte reprogramming into biliary progenitors upon injury. Together, these findings uncover molecular heterogeneity within the ductal epithelium and reveal YAP as a protective rheostat and regenerative regulator in the mammalian liver.

The majority of oligodendrocytes in the neocortex originate from neural progenitors that reside in the dorsal forebrain. We recently showed that Sonic Hedgehog (Shh) signaling in these dorsal progenitors is required to produce normal numbers of neocortical oligodendrocytes during embryonic development. Conditional deletion of the Shh signaling effector, Smo, in dorsal progenitors caused a dramatic reduction in oligodendrocyte numbers in the embryonic neocortex. In the current study, we show that the depleted oligodendrocyte lineage in Smo conditional mutants is able to recover to control numbers over time. This eventual recovery is achieved in part by expansion of the ventrally-derived wild-type lineage that normally makes up a minority of the total oligodendrocyte population. However, we find that the remaining dorsally-derived mutant cells also increase in numbers over time to contribute equally to the recovery of the total population. Additionally, we found that the ways in which the dorsal and ventral sources cooperate to achieve recovery is different for distinct populations of oligodendrocyte-lineage cells. Oligodendrocyte precursor cells (OPCs) in the neocortical white matter recover completely by expansion of the remaining dorsally-derived Smo mutant cells. On the other hand, mature oligodendrocytes in the white and gray matter recover through an equal contribution from dorsal mutant and ventral wild-type lineages. Interestingly, the only population that did not make a full recovery was OPCs in the gray matter. We find that gray matter OPCs are less proliferative in Smo cKO mutants compared to controls, which may explain their inability to fully recover. Our data indicate that certain populations of the dorsal oligodendrocyte lineage are more affected by loss of Shh signaling than others. Furthermore, these studies shed new light on the complex relationship between dorsal and ventral sources of oligodendrocytes in the developing neocortex.

From a whole macaque (m. fascicularis) brain, serial transversal sections of the brainstem were made using a blockface setup that takes high-definition pictures of the organ surface, before microtome slicing. Different histological techniques were applied to series of sections which allowed us to identify RTN neurons among Phox2b immunoreactive cells within the macaque brainstem. Our findings rely on the specific signature previously assessed in rats for chemosensitive neurons of the parafacial region: Phox2b/VGluT2/Galanin. Blockface photographs allowed us to correct histology-induced deformations on digitized sections. Series of sections were virtually stacked and ppGal/Phox2b positive cells identified to provide with the first 3D description of the main chemosensitive brainstem region in non-human primates.

Infiltration of immune cells into the tumor microenvironment (TME) can regulate growth and survival of neoplastic cells, impacting tumorigenesis and tumor progression. Correlations between the number of effector immune cells present in a tumor and clinical outcomes in many human tumors, including breast, have been widely described. Current immunotherapies utilizing checkpoint inhibitors or co-stimulatory molecule agonists aim to activate effector immune cells. However, tumors often lack adequate effector cell numbers within the TME, resulting in suboptimal responses to these agents. Chemerin (RARRES2) is a leukocyte chemoattractant widely expressed in many tissues and is known to recruit innate leukocytes. CMKLR1 is a chemotactic cellular receptor for chemerin and is expressed on subsets of dendritic cells, NK cells, and macrophages. We have previously shown that chemerin acts as a tumor suppressive cytokine in mouse melanoma models by recruiting innate immune defenses into the TME. Chemerin/RARRES2 is down-regulated in many tumors, including breast, compared to normal tissue counterparts. Here, using a syngeneic orthotopic EMT6 breast carcinoma model, we show that forced overexpression of chemerin by tumor cells results in significant recruitment of NK cells and T cells within the TME. While chemerin secretion by EMT6 cells did not alter their phenotypic behavior in vitro, it did significantly suppress tumor growth in vivo. To define the cellular effectors required for this anti-tumor phenotype, we depleted NK cells or CD8+ T cells and found that either cell type is required for chemerin-dependent suppression of EMT6 tumor growth. Finally, we show significantly reduced levels of RARRES2 mRNA in human breast cancer samples compared to matched normal tissues. Thus, for the first time we have shown that increasing chemerin expression within the breast carcinoma TME can suppress growth by recruitment of NK and T cells, thereby supporting this approach as a promising immunotherapeutic strategy.

The genetic programs responsible for pulmonary lymphatic maturation prior to birth are not known. To address this gap in knowledge, we developed a novel cell sorting strategy to collect fetal pulmonary lymphatic endothelial cells (PLECs) for global transcriptional profiling. We identified PLECs based on their unique cell surface immunophenotype (CD31+/Vegfr3+/Lyve1+/Pdpn+) and isolated them from murine lungs during late gestation (E16.5, E17.5, E18.5). Gene expression profiling was performed using whole-genome microarrays, and 1,281 genes were significantly differentially expressed with respect to time (FDR q < 0.05) and grouped into six clusters. Two clusters containing a total of 493 genes strongly upregulated at E18.5 were significantly enriched in genes with functional annotations corresponding to innate immune response, positive regulation of angiogenesis, complement & coagulation cascade, ECM/cell-adhesion, and lipid metabolism. Gene Set Enrichment Analysis identified several pathways coordinately upregulated during late gestation, the strongest of which was the type-I IFN-α/β signaling pathway. Upregulation of canonical interferon target genes was confirmed by qRT-PCR and in situ hybridization in E18.5 PLECs. We also identified transcriptional events consistent with a prenatal PLEC maturation program. This PLEC-specific program included individual genes (Ch25h, Itpkc, Pcdhac2 and S1pr3) as well as a set of chemokines and genes containing an NF-κB binding site in their promoter. Overall, this work reveals transcriptional insights into the genes, signaling pathways and biological processes associated with pulmonary lymphatic maturation in the fetal lung.

Melanoma is one of the most common skin malignancies. Both microRNAs and long non-coding RNAs (lncRNAs) have critical roles in the progression of cancers, including melanoma. However, the underlying molecular mechanism has not been fully characterized. We demonstrated that miR-34a is negatively correlated with MALAT1 in melanoma cells and tumor specimens. Interestingly, MALAT1, which contains functional sequence-specific miR-34a-binding sites, regulates miR-34a stability in melanoma cells and in vivo. Importantly, MALAT1 was significantly enriched in the Ago2 complex, but not when the MALAT1-binding site of miR-34a was mutated. Furthermore, MALAT1 could be shown to regulate c-Myc and Met expression by functioning as a miR-34a sponge. Our results reveal an unexpected mode of action for MALAT1 as an important regulator of miR-34a.

Acoustic signals are relayed from the ear to the brain via spiral ganglion neurons (SGNs) that receive auditory information from the cochlear inner hair cells and transmit that information to the cochlear nucleus of the brainstem. Physiologically distinct classes of SGNs have been characterized by their spontaneous firing rate and responses to sound, and those physiological distinctions are thought to correspond to stereotyped synaptic positions on the inner hair cell (IHC). More recently, single cell profiling has identified multiple groups of SGNs based on transcriptional profiling, however, correlations between any of these groups and distinct neuronal physiology has not been determined.In this study, we show that expression of the POU transcription factor Pou4f1 in Type I SGNs in mice of both sexes correlates with a synaptic location on the modiolar side of IHCs. Conditional deletion of Pou4f1 in SGNs beginning in mice at embryonic day 13 rescues the early pathfinding and apoptotic phenotypes reported for germline deletion of Pou4f1, resulting in a phenotypically normal development of SGN patterning. However, conditional deletion of Pou4f1 in SGNs alters the activation of Ca2+-channels in IHCs, primarily by increasing their voltage-sensitivity. Moreover, the modiolar to pillar gradient of active zone (AZ) Ca2+-influx strength is eliminated. These results demonstrate that a subset of modiolar-targeted SGNs retain expression of Pou4f1 beyond the onset of hearing and suggest that this transcription factor plays an instructive role in presynaptic Ca2+ signaling in IHCs.Significance:Physiologically distinct classes of Type I spiral ganglion neurons (SGNs) are necessary to encode sound intensities spanning the audible range. While anatomical studies have demonstrated structural correlates for some physiologically-defined classes of Type I SGNs, an understanding of the molecular pathways that specify each type is only emerging. Here, we demonstrate that expression of the transcription factor Pou4f1 corresponds to a distinct subgroup of Type I SGNs that synapse on the modiolar side of inner hair cells. The conditional deletion of Pou4f1 after SGN formation does not disrupt ganglion size or morphology, change the distribution of IHC synaptic locations, or impact the creation of synapses, but does influence the voltage-dependence and strength of Ca2+-influx at presynaptic AZs in IHCs.

Parvalbumin (PV) expressing GABAergic interneurons provide large source of GABA to spiny projection neurons (SPNs) in the striatum. However, the roles of PV+ interneurons in the regulation of SPNs in the ventral striatum and emotional states are largely unknown. Here, we investigated whether stimulation of ventral striatal (accumbal) PV+ interneurons would drive emotional valence in mice. We found that during conditioned place preference (CPP) training, activation of accumbal PV+ interneurons evoked place preference while suppressing them resulted in conditioned place aversion (CPA). Activation of PV+interneurons during place conditioning increased Fos expression in SPNs in the direct pathway (dSPNs) and impaired lithium chloride-induced CPA. Activation of dSPNs and SPNs in the indirect pathway (iSPNs) induced CPP and CPA, respectively; conversely, suppression of dSPNs or iSPNs induced CPA or CPP. In addition, activation or suppression of calretinin-expressing (CR) GABAergic interneurons did not induce place preference or aversion. These data suggest that PV+ interneurons can bidirectionally determine the emotional valence through their regulation of accumbal SPN activities and raise the possibility that manipulation of PV+ interneuron activity may have the potential to alter emotional valence and treat related mental disorders.

Triple-negative breast cancers (TNBCs) represent a heterogeneous disease characterized by several molecular subtypes with different prognoses and responses to therapy. For a correct clinical management of TNBC patients the knowledge of the gene regulation mechanisms related to tumor progression and drug response has become fundamental.

LncRNAs regulate gene expression through various processes, including chromatin modification, transcription and post-transcription and they are emerging as important cancer biomarkers being involved in tumor pathogenesis, metastatic progression and drug resistance.

In this study we aimed to analyze the expression of the lncRNA HOTAIR, mainly involved in breast cancer disease, in a large case series of TNBC patients. We used ISH methods by a RNA probe to better define its staining in tumor tissues and its relation with clinical-pathological parameters and outcomes of patients.

Our results show that high HOTAIR expression in tumor tissues is strongly correlated with lymph nodes metastasis (LNM) (p=0.039), as reported also for other tumor types, and has a direct strong association with Androgen Receptor (AR) expression (p= 0.019).

These data confirm the prognostic role of HOTAIR in TNBC, and, its involvement in the regulation of AR pathway, suggests the possibility to establish new therapeutic strategies for AR+TNBC patients.

Abstract

OBJECTIVE:

Cancer-associated fibroblasts (CAFs), a major component of cancer stroma, can confer aggressive properties to cancer cells by secreting multiple factors. Their phenotypes are stably maintained, but the mechanisms are not fully understood. We aimed to show the critical role of epigenetic changes in CAFs in maintaining their tumour-promoting capacity and to show the validity of the epigenomic approach in identifying therapeutic targets from CAFs to starve cancer cells.

DESIGN:

Twelve pairs of primary gastric CAFs and their corresponding non-CAFs (NCAFs) were established from surgical specimens. Genome-wide DNA methylation and H3K27me3 analyses were conducted by BeadArray 450K and ChIP-on-Chip, respectively. Functions of potential a therapeutic target were analysed by inhibiting it, and prognostic impact was assessed in a database.

RESULTS:

CAFs had diverse and distinct DNA methylation and H3K27me3 patterns compared with NCAFs. Loss of H3K27me3, but not DNA methylation, in CAFs was enriched for genes involved in stem cell niche, cell growth, tissue development and stromal-epithelial interactions, such as WNT5A, GREM1, NOG and IGF2. Among these, we revealed that WNT5A, which had been considered to be derived from cancer cells, was highly expressed in cancer stromal fibroblasts, and was associated with poor prognosis. Inhibition of secreted WNT5A from CAFs suppressed cancer cell growth and migration.

CONCLUSIONS:

H3K27me3 plays a crucial role in defining tumour-promoting capacities of CAFs, and multiple stem cell niche factors were secreted from CAFs due to loss of H3K27me3. The validity of the epigenetic approach to uncover therapeutic targets for cancer-starving therapy was demonstrated.

The dorsomedial striatum (DMS) is critically involved in motor control and reward processing, but the specific neural circuit mediators are poorly understood. Recent evidence highlights the extensive connectivity of low-threshold spiking interneurons (LTSIs) within local striatal circuitry; however, the in vivo function of LTSIs remains largely unexplored. We employed fiber photometry to assess LTSI calcium activity in a range of DMS-mediated behaviors, uncovering specific reward-related activity that is down-modulated during goal-directed learning. Using two mechanistically distinct manipulations, we demonstrated that this down-modulation of LTSI activity is critical for acquisition of novel contingencies, but not for their modification. In contrast, continued LTSI activation slowed instrumental learning. Similar manipulations of fast-spiking interneurons did not reproduce these effects, implying a specific function of LTSIs. Finally, we revealed a role for the γ-aminobutyric acid (GABA)ergic functions of LTSIs in learning. Together, our data provide new insights into this striatal interneuron subclass as important gatekeepers of goal-directed learning.