Lhx1 functions together with Otx2, Foxa2, and Ldb1 to govern anterior mesendoderm, node, and midline development.

Gene regulatory networks controlling functional activities of spatially and temporally distinct endodermal cell populations in the early mouse embryo remain ill defined. The T-box transcription factor Eomes, acting downstream from Nodal/Smad signals, directly activates the LIM domain homeobox transcription factor Lhx1 in the visceral endoderm. Here we demonstrate Smad4/Eomes-dependent Lhx1 expression in the epiblast marks the entire definitive endoderm lineage, the anterior mesendoderm, and midline progenitors. Conditional inactivation of Lhx1 disrupts anterior definitive endoderm development and impedes node and midline morphogenesis in part due to severe disturbances in visceral endoderm displacement. Transcriptional profiling and ChIP-seq (chromatin immunoprecipitation [ChIP] followed by high-throughput sequencing) experiments identified Lhx1 target genes, including numerous anterior definitive endoderm markers and components of the Wnt signaling pathway. Interestingly, Lhx1-binding sites were enriched at enhancers, including the Nodal-proximal epiblast enhancer element and enhancer regions controlling Otx2 and Foxa2 expression. Moreover, in proteomic experiments, we characterized a complex comprised of Lhx1, Otx2, and Foxa2 as well as the chromatin-looping protein Ldb1. These partnerships cooperatively regulate development of the anterior mesendoderm, node, and midline cell populations responsible for establishment of the left-right body axis and head formation.

Long read sequencing reveals novel isoforms and insights into splicing regulation during cell state changes.

BACKGROUND: Alternative splicing is a key mechanism underlying cellular differentiation and a driver of complexity in mammalian neuronal tissues. However, understanding of which isoforms are differentially used or expressed and how this affects cellular differentiation remains unclear. Long read sequencing allows full-length transcript recovery and quantification, enabling transcript-level analysis of alternative splicing processes and how these change with cell state. Here, we utilise Oxford Nanopore Technologies sequencing to produce a custom annotation of a well-studied human neuroblastoma cell line SH-SY5Y, and to characterise isoform expression and usage across differentiation. RESULTS: We identify many previously unannotated features, including a novel transcript of the voltage-gated calcium channel subunit gene, CACNA2D2. We show differential expression and usage of transcripts during differentiation identifying candidates for future research into state change regulation. CONCLUSIONS: Our work highlights the potential of long read sequencing to uncover previously unknown transcript diversity and mechanisms influencing alternative splicing.

Cellular calcium in bipolar disorder: systematic review and meta-analysis.

Calcium signalling has long been implicated in bipolar disorder, especially by reports of altered intracellular calcium ion concentrations ([Ca2+]). However, the evidence has not been appraised critically. We carried out a systematic review and meta-analysis of studies of cellular calcium indices in bipolar disorder. 2281 records were identified and 117 screened, of which 32 were eligible and 21 were suitable for meta-analyses. The latter each involved up to 642 patients and 404 control subjects. We found that basal free intracellular [Ca2+] is increased in bipolar disorder, both in platelets and in lymphocytes. The effect size is 0.55, with an estimated elevation of 29%. It is observed in medication-free patients. It is present in mania and bipolar depression, but data are equivocal for euthymia. Cells from bipolar disorder individuals also show an enhanced [Ca2+] response to stimulation with 5-HT or thrombin, by an estimated 25%, with an effect size of 0.63. In studies which included other diagnoses, intracellular basal [Ca2+] was higher in bipolar disorder than in unipolar depression, but not significantly different from schizophrenia. Functional parameters of cellular Ca2+ (e.g. calcium transients), and neuronal [Ca2+], have been much less investigated, and no firm conclusions can be drawn. In summary, there is a robust, medium effect size elevation of basal and stimulated free intracellular [Ca2+] in bipolar disorder. The results suggest altered calcium functioning in the disorder, and encourage further investigations into the underlying mechanisms, and the implications for pathophysiology and therapeutics.

Blimp1/Prdm1 governs terminal differentiation of endovascular trophoblast giant cells and defines multipotent progenitors in the developing placenta.

Developmental arrest of Blimp1/Prdm1 mutant embryos at around embryonic day 10.5 (E10.5) has been attributed to placental disturbances. Here we investigate Blimp1/Prdm1 requirements in the trophoblast cell lineage. Loss of function disrupts specification of the invasive spiral artery-associated trophoblast giant cells (SpA-TGCs) surrounding maternal blood vessels and severely compromises the ability of the spongiotrophoblast layer to expand appropriately, secondarily causing collapse of the underlying labyrinth layer. Additionally, we identify a population of proliferating Blimp1(+) diploid cells present within the spongiotrophoblast layer. Lineage tracing experiments exploiting a novel Prdm1.Cre-LacZ allele demonstrate that these Blimp1(+) cells give rise to the mature SpA-TGCs, canal TGCs, and glycogen trophoblasts. In sum, the transcriptional repressor Blimp1/Prdm1 is required for terminal differentiation of SpA-TGCs and defines a lineage-restricted progenitor cell population contributing to placental growth and morphogenesis.

The transcriptional repressor Blimp1 is expressed in rare luminal progenitors and is essential for mammary gland development.

Mammary gland morphogenesis depends on a tight balance between cell proliferation, differentiation and apoptosis, to create a defined functional hierarchy within the epithelia. The limited availability of stem cell/progenitor markers has made it challenging to decipher lineage relationships. Here, we identify a rare subset of luminal progenitors that express the zinc finger transcriptional repressor Blimp1, and demonstrate that this subset of highly clonogenic luminal progenitors is required for mammary gland development. Conditional inactivation experiments using K14-Cre and WAPi-Cre deleter strains revealed essential functions at multiple developmental stages. Thus, Blimp1 regulates proliferation, apoptosis and alveolar cell maturation during puberty and pregnancy. Loss of Blimp1 disrupts epithelial architecture and lumen formation both in vivo and in three-dimensional (3D) primary cell cultures. Collectively, these results demonstrate that Blimp1 is required to maintain a highly proliferative luminal subset necessary for mammary gland development and homeostasis.

Blimp1/Prdm1 Functions in Opposition to Irf1 to Maintain Neonatal Tolerance during Postnatal Intestinal Maturation.

The neonatal intestine is a very complex and dynamic organ that must rapidly adapt and remodel in response to a barrage of environmental stimuli during the first few postnatal weeks. Recent studies demonstrate that the zinc finger transcriptional repressor Blimp1/Prdm1 plays an essential role governing postnatal reprogramming of intestinal enterocytes during this period. Functional loss results in global changes in gene expression patterns, particularly in genes associated with metabolic function. Here we engineered a knock-in allele expressing an eGFP-tagged fusion protein under control of the endogenous regulatory elements and performed genome wide ChIP-seq analysis to identify direct Blimp1 targets and further elucidate the function of Blimp1 in intestinal development. Comparison with published human and mouse datasets revealed a highly conserved core set of genes including interferon-inducible promoters. Here we show that the interferon-inducible transcriptional activator Irf1 is constitutively expressed throughout fetal and postnatal intestinal epithelium development. ChIP-seq demonstrates closely overlapping Blimp1 and Irf1 peaks at key components of the MHC class I pathway in fetal enterocytes. The onset of MHC class I expression coincides with down-regulated Blimp1 expression during the suckling to weaning transition. Collectively, these experiments strongly suggest that in addition to regulating the enterocyte metabolic switch, Blimp1 functions as a gatekeeper in opposition to Irf1 to prevent premature activation of the MHC class I pathway in villus epithelium to maintain tolerance in the neonatal intestine.

The transcriptional repressor Blimp1/Prdm1 regulates postnatal reprogramming of intestinal enterocytes.

Female mammals produce milk to feed their newborn offspring before teeth develop and permit the consumption of solid food. Intestinal enterocytes dramatically alter their biochemical signature during the suckling-to-weaning transition. The transcriptional repressor Blimp1 is strongly expressed in immature enterocytes in utero, but these are gradually replaced by Blimp1(-) crypt-derived adult enterocytes. Here we used a conditional inactivation strategy to eliminate Blimp1 function in the developing intestinal epithelium. There was no noticeable effect on gross morphology or formation of mature cell types before birth. However, survival of mutant neonates was severely compromised. Transcriptional profiling experiments reveal global changes in gene expression patterns. Key components of the adult enterocyte biochemical signature were substantially and prematurely activated. In contrast, those required for processing maternal milk were markedly reduced. Thus, we conclude Blimp1 governs the developmental switch responsible for postnatal intestinal maturation.

Menin and p53 have non-synergistic effects on tumorigenesis in mice.

BACKGROUND: While it is now more than a decade since the first description of the gene mutation underlying the tumour predisposition syndrome multiple endocrine neoplasia type 1 (MEN1), the mechanism by which its protein product menin acts to prevent development of tumours is still poorly understood. METHODS: We undertook a genetic experiment to assess whether menin synergises with p53. Mice carrying various combinations of Men1 and Trp53 mutations were generated then survival and pathology assessed. RESULTS: While homozygous loss of Trp53 in mice resulted in early onset, aggressive tumours and profoundly reduced lifespan, heterozygous loss of either Trp53 or Men1 caused later onset disease, with a spectrum of tumours characteristic of each tumour suppressor gene. Loss of one copy of Men1 in animals also lacking both alleles of Trp53 did not exacerbate phenotype, based on survival, animal weight or sites of pathology, compared to Trp53 deletion alone. Dual heterozygous deletion of Men1 and Trp53 resulted in a small reduction in lifespan compared to the individual mutations, without new tumour sites. In the adrenal, we observed development of cortical tumours in dual heterozygous animals, as we have previously seen in Men1+/- animals, and there was loss of heterozygosity at the Men1 allele in these tumours. Median number of pathology observations per animal was increased in dual heterozygous animals compared with heterozygous loss of Trp53 alone. CONCLUSIONS: Simultaneous heterozygous deletion of Men1 in animals with either heterozygous or homozygous deletion of Trp53 did not result in formation of tumours at any new sites, implying additive rather than synergistic effects of these pathways. Mice that were Men1+/- in addition to Trp53+/- had tumours in endocrine as well as other sites, implying that increase in total tumour burden, at sites typically associated with either Men1 or Trp53 loss, contributed to the slight decrease in survival in Men1+/-: Trp53+/- animals in comparison with their littermates.

New drug targets in psychiatry: Neurobiological considerations in the genomics era.

After a period of withdrawal, pharmaceutical companies have begun to reinvest in neuropsychiatric disorders, due to improvements in our understanding of these disorders, stimulated in part by genomic studies. However, translating this information into disease insights and ultimately into tractable therapeutic targets is a major challenge. Here we consider how different sources of information might be integrated to guide this process. We review how an understanding of neurobiology has been used to advance therapeutic candidates identified in the pre-genomic era, using catechol-O-methyltransferase (COMT) as an exemplar. We then contrast with ZNF804A, the first genome-wide significant schizophrenia gene, and draw on some of the lessons that these and other examples provide. We highlight that, at least in the short term, the translation of potential targets for which there is orthogonal neurobiological support is likely to be more straightforward and productive than that those relying solely on genomic information. Although we focus here on information from genomic studies of schizophrenia, the points are broadly applicable across major psychiatric disorders and their symptoms.

Kalirin as a Novel Treatment Target for Cognitive Dysfunction in Schizophrenia.

The cognitive dysfunction experienced by patients with schizophrenia represents a major unmet clinical need. We believe that enhancing synaptic function and plasticity by targeting kalirin may provide a novel means to remediate these symptoms. Karilin (a protein encoded by the KALRN gene) has multiple functional domains, including two Dbl homology (DH) guanine exchange factor (GEF) domains, which act to enhance the activity of the Rho family guanosine triphosphate (GTP)-ases. Here, we provide an overview of kalirin's roles in brain function and its therapeutic potential in schizophrenia. We outline how it mediates diverse effects via a suite of distinct isoforms that couple to members of the Rho GTPase family to regulate synapse formation and stabilisation, and how genomic and post-mortem data implicate it in schizophrenia. We then review the current state of knowledge about the influence of kalirin on brain function at a systems level, based largely on evidence from transgenic mouse models, which support its proposed role in regulating dendritic spine function and plasticity. We demonstrate that, whilst the GTPases are classically considered to be 'undruggable', targeting kalirin and other Rho GEFs provides a means to indirectly modulate their activity. Finally, we integrate across the information presented to assess the therapeutic potential of kalirin for schizophrenia and highlight the key outstanding questions required to advance it in this capacity; namely, the need for more information about the diversity and function of its isoforms, how these change across neurodevelopment, and how they affect brain function in vivo.

CACNA1C (CaV1.2) and other L-type calcium channels in the pathophysiology and treatment of psychiatric disorders: Advances from functional genomics and pharmacoepidemiology.

A role for voltage-gated calcium channels (VGCCs) in psychiatric disorders has long been postulated as part of a broader involvement of intracellular calcium signalling. However, the data were inconclusive and hard to interpret. We review three areas of research that have markedly advanced the field. First, there is now robust genomic evidence that common variants in VGCC subunit genes, notably CACNA1C which encodes the L-type calcium channel (LTCC) CaV1.2 subunit, are trans-diagnostically associated with psychiatric disorders including schizophrenia and bipolar disorder. Rare variants in these genes also contribute to the risk. Second, pharmacoepidemiological evidence supports the possibility that calcium channel blockers, which target LTCCs, might have beneficial effects on the onset or course of these disorders. This is especially true for calcium channel blockers that are brain penetrant. Third, long-range sequencing is revealing the repertoire of full-length LTCC transcript isoforms. Many novel and abundant CACNA1C isoforms have been identified in human and mouse brain, including some which are enriched compared to heart or aorta, and predicted to encode channels with differing functional and pharmacological properties. These isoforms may contribute to the molecular mechanisms of genetic association to psychiatric disorders. They may also enable development of therapeutic agents that can preferentially target brain LTCC isoforms and be of potential value for psychiatric indications.

Targeting synaptic plasticity in schizophrenia: insights from genomic studies.

Patients with schizophrenia experience cognitive dysfunction and negative symptoms that do not respond to current drug treatments. Historical evidence is consistent with the hypothesis that these deficits are due, at least in part, to altered cortical synaptic plasticity (the ability of synapses to strengthen or weaken their activity), making this an attractive pathway for therapeutic intervention. However, while synaptic transmission and plasticity is well understood in model systems, it has been challenging to identify specific therapeutic targets for schizophrenia. New information is emerging from genomic findings, which converge on synaptic plasticity and provide a new window on the neurobiology of schizophrenia. Translating this information into therapeutic advances will require a multidisciplinary and collaborative approach.

The transcriptional repressor Blimp1/PRDM1 regulates the maternal decidual response in mice.

The transcriptional repressor Blimp1 controls cell fate decisions in the developing embryo and adult tissues. Here we describe Blimp1 expression and functional requirements within maternal uterine tissues during pregnancy. Expression is robustly up-regulated at early post-implantation stages in the primary decidual zone (PDZ) surrounding the embryo. Conditional inactivation results in defective formation of the PDZ barrier and abnormal trophectoderm invasion. RNA-Seq analysis demonstrates down-regulated expression of genes involved in cell adhesion and markers of decidualisation. In contrast, genes controlling immune responses including IFNγ are up-regulated. ChIP-Seq experiments identify candidate targets unique to the decidua as well as those shared across diverse cell types including a highly conserved peak at the Csf-1 gene promoter. Interestingly Blimp1 inactivation results in up-regulated Csf1 expression and macrophage recruitment into maternal decidual tissues. These results identify Blimp1 as a critical regulator of tissue remodelling and maternal tolerance during early stages of pregnancy.

Global expression profiling of sex cord stromal tumors from Men1 heterozygous mice identifies altered TGF-beta signaling, decreased Gata6 and increased Csf1r expression.

Heterozygous disruption of the Men1 gene predisposes mice to the development of multiple endocrine tumors, accurately mimicking the human MEN1 cancer predisposition syndrome. Additionally, Men1(+/-) mice frequently develop sex cord adenomas. The mechanism underlying the susceptibility of these mice to sex cord tumor development has not been fully determined, but data suggest it may involve transcriptional regulation of key growth promoting/repressing genes. To identify potential menin-regulated genes that may be important for tumor suppression in sex cord cells, we compared the global gene expression profiles of testis and ovary adenomas with other endocrine tumors of the pancreas and pituitary from Men1 heterozygous mice and with control tissues. Gonadal tumors clustered separately from pancreas and pituitary tumors with only a few genes (e.g., Cdkn2c) commonly dysregulated in all tumor types. Testis and ovary tumors displayed a higher level of transcriptional similarity to each other than they did to their respective control tissues. Among genes that had decreased expression in tumors was significant over-representation of genes associated with the TGF-beta, hedgehog and Wnt signaling, indicating that loss of menin function affects these pathways at the level of transcription. Aberrant protein expression in Leydig and granulosa cells of 2 transcriptionally dysregulated gene products, Gata6 and Csf1r were confirmed by immunohistochemistry. We propose that sex cord tumor susceptibility in Men1(+/-) mice involves deregulated cell proliferation due to dysregulation of multiple cell growth regulating genes including: reduced Cdkn2c transcription, loss of TGF-beta pathway tumor suppressor function (e.g., Gata6) and transcriptional activation of Csf1r.

Identification of ARHGEF17, DENND2D, FGFR3, and RB1 mutations in melanoma by inhibition of nonsense-mediated mRNA decay.

Gene identification by nonsense-mediated mRNA decay inhibition (GINI) has proven to be a strategy for genome-wide discovery of genes containing inactivating mutations in colon and prostate cancers. Here, we present the first study of inhibition of the nonsense-mediated mRNA decay (NMD) pathway in melanoma. We used a combination of emetine and actinomycin D treatment to stabilize mRNAs containing premature termination codons (PTCs), followed by microarray analysis and sequencing to identify novel tumor suppressor genes (TSGs) in a panel of 12 melanoma cell lines. Stringent analysis of the array data was used to select 35 candidate genes for sequencing. Of these, 4 (11%) were found to carry PTCs, including ARHGEF17, DENND2D, FGFR3, and RB1. While RB1 mutations have previously been described in melanoma, the other three genes represent potentially novel melanoma; TSGs. ARHGEF17 showed a G1865A mutation leading to W622X in a cell line derived from a mucosal melanoma; in RB1 a C1411T base change resulting in Q471X was discovered in a cell line derived from an acral melanoma; and the FGFR3 and DENND2D genes had intronic insertions leading to PTCs in cell lines derived from superficially spreading melanomas. We conclude that although the false positive rate is high, most likely due to the lack of DNA mismatch repair gene defects, the GINI protocol is one approach to discover novel TSGs in melanoma.

The Oxford study of Calcium channel Antagonism, Cognition, Mood instability and Sleep (OxCaMS): study protocol for a randomised controlled, experimental medicine study.

BACKGROUND: The discovery that voltage-gated calcium channel genes such as CACNA1C are part of the aetiology of psychiatric disorders has rekindled interest in the therapeutic potential of L-type calcium channel (LTCC) antagonists. These drugs, licensed to treat hypertension and angina, have previously been used in bipolar disorder, but without clear results. Neither is much known about the broader effects of these drugs on the brain and behaviour. METHODS: The Oxford study of Calcium channel Antagonism, Cognition, Mood instability and Sleep (OxCaMS) is a high-intensity randomised, double-blind, placebo-controlled experimental medicine study on the effect of the LTCC antagonist nicardipine in healthy young adults with mood instability. An array of cognitive, psychiatric, circadian, physiological, biochemical and neuroimaging (functional magnetic resonance imaging and magnetoencephalography) parameters are measured during a 4-week period, with randomisation to drug or placebo on day 14. We are interested in whether nicardipine affects the stability of these measures, as well as its overall effects. Participants are genotyped for the CACNA1C risk polymorphism rs1006737. DISCUSSION: The results will clarify the potential of LTCC antagonists for repurposing or modification for use in psychiatric disorders in which cognition, mood and sleep are affected. TRIAL REGISTRATION: ISRCTN, ISRCTN33631053 . Retrospectively registered on 8 June 2018 (applied 17 May 2018).

Publisher Correction: Single-cell RNA-seq reveals cell type-specific transcriptional signatures at the maternal-foetal interface during pregnancy.

This corrects the article DOI: 10.1038/ncomms11414.

Blimp-1/PRDM1 is a critical regulator of Type III Interferon responses in mammary epithelial cells.

The transcriptional repressor Blimp-1 originally cloned as a silencer of type I interferon (IFN)-ß gene expression controls cell fate decisions in multiple tissue contexts. Conditional inactivation in the mammary gland was recently shown to disrupt epithelial cell architecture. Here we report that Blimp-1 regulates expression of viral defense, IFN signaling and MHC class I pathways, and directly targets the transcriptional activator Stat1. Blimp-1 functional loss in 3D cultures of mammary epithelial cells (MECs) results in accumulation of dsRNA and expression of type III IFN-λ. Cultures treated with IFN lambda similarly display defective lumen formation. These results demonstrate that type III IFN-λ profoundly influences the behavior of MECs and identify Blimp-1 as a critical regulator of IFN signaling cascades.

Combinatorial Smad2/3 Activities Downstream of Nodal Signaling Maintain Embryonic/Extra-Embryonic Cell Identities during Lineage Priming.

Epiblast cells in the early post-implantation stage mammalian embryo undergo a transition described as lineage priming before cell fate allocation, but signaling pathways acting upstream remain ill defined. Genetic studies demonstrate that Smad2/3 double-mutant mouse embryos die shortly after implantation. To learn more about the molecular disturbances underlying this abrupt failure, here we characterized Smad2/3-deficient embryonic stem cells (ESCs). We found that Smad2/3 double-knockout ESCs induced to form epiblast-like cells (EpiLCs) display changes in naive and primed pluripotency marker gene expression, associated with the disruption of Oct4-bound distal regulatory elements. In the absence of Smad2/3, we observed enhanced Bmp target gene expression and de-repression of extra-embryonic gene expression. Cell fate allocation into all three embryonic germ layers is disrupted. Collectively, these experiments demonstrate that combinatorial Smad2/3 functional activities are required to maintain distinct embryonic and/or extra-embryonic cell identity during lineage priming in the epiblast before gastrulation.